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Sample records for acrosome reacted sperm

  1. Induction of acrosome reactions of canine sperm by homologous zona pellucida.

    PubMed

    Kawakami, E; Vandevoort, C A; Mahi-Brown, C A; Overstreet, J W

    1993-04-01

    In this study the induction of the acrosome reaction of canine sperm by homologous zona pellucida (ZP) was examined. Twelve semen samples obtained from 6 normal beagle dogs were evaluated after sperm incubation in vitro with canine capacitation medium (CCM). Washed sperm were preincubated at 37 degrees C in 5% CO2 in air for 4 and 7 h prior to experimental treatment. Sperm were co-incubated for 1 min with intact oocytes collected from canine ovaries. Half of the oocytes were then fixed, and the bound sperm were assessed for acrosome reactions through use of a polyclonal antisperm antiserum and indirect immunofluorescence. The remaining oocytes were incubated in sperm-free medium for an additional 1-h period, and the acrosomal status of sperm bound to the ZP was evaluated similarly. The percentage of acrosome-reacted sperm on the ZP increased significantly during the 1-h incubation period. In other experiments, capacitated canine sperm were incubated with heat-solubilized ZP for 1 h and their acrosomal status was determined using fluoresceinated Pisum sativum lectin. The percentages of acrosome-reacted sperm increased significantly in ZP solution compared with controls. These data demonstrate that intact and solubilized canine ZP are capable of inducing acrosome reactions of canine sperm.

  2. Kinetics of human sperm acrosomal exocytosis.

    PubMed

    Sosa, C M; Pavarotti, M A; Zanetti, M N; Zoppino, F C M; De Blas, G A; Mayorga, L S

    2015-03-01

    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.

  3. Evaluation of sperm acrosome reaction in the Asiatic elephant.

    PubMed

    Kitiyanant, Y; Schmidt, M J; Pavasuthipaisit, K

    2000-03-01

    This study focuses on the effect of chemicals on acrosome reaction in elephant spermatozoa. Semen was collected at the Washington Park Zoo in Portland, Oregon, from an 11-yr-old Asian elephant by artificial vagina (7 ejaculates) and transported to Mahidol University in Bangkok in extender at 4 to 5 degrees C within 24 to 28 h. A total of 500 x 10(6) sperm/mL was used for the control and for each of the 4 treatment groups: 1) cAMP (0.1 mM); 2) caffeine (0.1 mM); 3) Penicillamine hypotaurine and epinephine, PHE (penicillamine 2 mM, hypotaurine 1 mM, epinephine 1 mM); and 4) heparin (10 microg/mL) at 39 degrees C for 2 h. Aliquots were removed and the sperm viability, abnormal morphology, and acrosome status were evaluated by triple stain technique. Transmission electron microscopy (TEM) was used to observe changes of the sperm head membrane in all treatment groups. Trypan blue reliably stained dead spermatozoa, while rose Bengal stained only the spermatozoa with intact acrosomes. The concentration of dead sperm cells was similar in the 4 groups. The percentages of live acrosome-reacted spermatozoa in the control and in groups treated with caffeine, PHE, cAMP and heparin were 19.5 +/- 4.3, 38.1 +/- 4.0, 34.8 +/- 3.7, 29.8 +/- 0.8 and 28.0 +/- 4.2, respectively. The acrosome reaction rate was higher in the treatment groups than in the control (P<0.05). Caffeine and PHE caused significantly higher acrosome reaction of the sperm head than cAMP or heparin (P<0.05). The electron micrographs showed that the acrosome reaction occurred by the presence of apical vesiculation. The results indicated that 1) the triple stain technique allowed for evaluation of both viability and acrosome reaction simultaneously in elephant spermatozoa,2) acrosome reaction occurred at a high rate in all 3 treatment groups. 3) the effects of caffeine and PHE were significantly higher (P<0.05) than of cAMP and heparin, and 4) the data obtained from the triple stain technique corresponded to those

  4. Sea urchin sperm antigens mediating the acrosome reaction

    SciTech Connect

    Trimmer, J.S.

    1987-01-01

    The study of sea urchin sperm antigens mediating the acrosome reactions (AR) has been undertaken. Monoclonal antibodies (mAbs) have been isolated reacting with a number of sperm surface antigens. These mAbs have been used in functional assays to attempt to infer the roles of these proteins in the induction of the AR. These mAbs have also been used to isolate protein for biochemical characterization and reconstitution studies. mAbs reacting with a 210 kD protein of the sea urchin sperm plasma membrane have been used to identify this protein as playing a role in the regulation of ion fluxes during the induction of the AR. mAbs reacting with certain extracellular regions inhibit the induction of: the AR, the long duration {sup 45}Ca{sup 2+} uptake into the mitochondrion, and H{sup +} efflux. Addition of these same mAbs, however, induces an increase in sperm (Ca{sup 2+}){sub i} to levels much higher than those induced by FSG, as monitored by the fluorescent Ca{sup 2+} indicators fura 2 and indo 1. This (Ca{sup 2+}){sub i} increase occurs without an increase in pH{sub i}, and thus allows for the first time the analysis of the effects of increasing sperm (Ca{sup 2+}){sub i} ion the absence of increased pH{sub i}.

  5. Differential release of guinea pig sperm acrosomal components during exocytosis.

    PubMed

    Kim, K S; Foster, J A; Gerton, G L

    2001-01-01

    The contents of the sperm acrosome are compartmentalized at the biochemical and morphological levels. Biochemically, the acrosome can be considered to be comprised of two compartments: one consisting of readily soluble proteins and one containing a particulate acrosomal matrix. To test the hypothesis that compartmentalization affects the release of acrosomal components during the course of secretion in guinea pig sperm, we examined the relationship between the presence of specific proteins and acrosomal status and monitored the recovery of acrosomal constituents in the medium surrounding sperm induced to undergo exocytosis with the ionophore A23187. Cysteine-rich secretory protein 2 (CRISP-2), a soluble component of the acrosome, was rapidly lost from the acrosome soon after ionophore treatment. However, acrosomal matrix components remained associated with the sperm for longer periods. AM67, a matrix component and the guinea pig orthologue of the mouse sperm zona pellucida-binding protein sp56, was released at a slower rate than was CRISP-2 but at a faster rate than were two other matrix proteins, AM50 and proacrosin. Coincident with their release from the sperm, AM50 and proacrosin were posttranslationally modified, probably by proteolysis. The release of proacrosin from the matrix appears associated with the conversion of this protein to the enzymatically active acrosin protease. These results provide strong support for the hypothesis that compartmentalization plays a significant role in regulating the release of proteins during the course of acrosomal exocytosis. Acrosomal matrix proteins remain associated with the sperm for prolonged periods of time following the induction of acrosomal exocytosis, suggesting that transitional acrosomal intermediates may have significant functions in the fertilization process.

  6. Remodeling of the plasma membrane in preparation for sperm-egg recognition: roles of acrosomal proteins.

    PubMed

    Tanphaichitr, Nongnuj; Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark

    2015-01-01

    The interaction of sperm with the egg's extracellular matrix, the zona pellucida (ZP) is the first step of the union between male and female gametes. The molecular mechanisms of this process have been studied for the past six decades with the results obtained being both interesting and confusing. In this article, we describe our recent work, which attempts to address two lines of questions from previous studies. First, because there are numerous ZP binding proteins reported by various researchers, how do these proteins act together in sperm-ZP interaction? Second, why do a number of acrosomal proteins have ZP affinity? Are they involved mainly in the initial sperm-ZP binding or rather in anchoring acrosome reacting/reacted spermatozoa to the ZP? Our studies reveal that a number of ZP binding proteins and chaperones, extracted from the anterior sperm head plasma membrane, coexist as high molecular weight (HMW) complexes, and that these complexes in capacitated spermatozoa have preferential ability to bind to the ZP. Zonadhesin (ZAN), known as an acrosomal protein with ZP affinity, is one of these proteins in the HMW complexes. Immunoprecipitation indicates that ZAN interacts with other acrosomal proteins, proacrosin/acrosin and sp32 (ACRBP), also present in the HMW complexes. Immunodetection of ZAN and proacrosin/acrosin on spermatozoa further indicates that both proteins traffic to the sperm head surface during capacitation where the sperm acrosomal matrix is still intact, and therefore they are likely involved in the initial sperm-ZP binding step.

  7. Identification of a ZP3-binding protein on acrosome-intact mouse sperm by photoaffinity crosslinking

    SciTech Connect

    Bleil, J.D.; Wassarman, P.M. )

    1990-07-01

    During the process of fertilization in mammals, sperm bind in a relatively species-specific manner to the zona pellucida (ZP) of ovulated eggs. ZP3, a glycoprotein found in the mouse egg zona pellucida, serves as receptor for sperm during gamete adhesion. We report here that a Mr 56,000 protein found on mouse sperm has properties expected for a sperm component that recognizes and binds to ZP3. This sperm protein is radiolabeled preferentially by a photoactivatable heterobifunctional crosslinker (Denny-Jaffee reagent) covalently linked to purified ZP3, binds very tightly to ZP3-affinity columns, and is localized to heads of acrosome-intact but not acrosome-reacted sperm. These and other findings suggest that this protein may be a ZP3-binding protein that, together with the sperm receptor, supports species-specific binding of mouse sperm to unfertilized eggs.

  8. Hydrogen peroxide induces premature acrosome reaction in rat sperm and reduces their penetration of the zona pellucida.

    PubMed

    Hsu, P C; Hsu, C C; Guo, Y L

    1999-11-29

    Recent studies have demonstrated that mammalian sperm are capable of generating reactive oxygen species (ROS) and that this activity is significantly accelerated in subfertile subjects. The observed decrease in penetration of zona-intact oocyte might be explained by chemical-induced ROS-related early onset of capacitation and premature acrosome reaction, but the mechanism is not clear. We determine whether zona-intact oocyte penetration capability in rat epididymal sperm was affected by premature acrosome reaction in rat sperm treated with hydrogen peroxide (H2O2) and calcium ionophore A23187 or H2O2 and lysophosphatidyl choline. Chlortetracycline fluorescence assay was used to study the status of acrosome reaction on epididymal sperm. The sperm-oocyte binding and penetration assay was used to evaluate the capability for zona pellucida penetration. There was a positive linear correlation between the frequency of acrosome-reacted sperm and capability of sperm-oocyte binding and penetration in zona-free oocytes. In the zona-intact oocytes, the sperm-oocyte penetration rate was suppressed as the proportions of acrosome-reacted sperm increased. In summary, this study showed that premature acrosome reaction reduced rat sperm's capability of penetrating zona-intact oocytes. However, this reduction is not seen in zona-free oocytes. These findings may provide a basis for understanding the effects of sperm ROS generation on zona pellucida penetration in male reproductive toxicology.

  9. Inositol 1,4,5-trisphosphate receptors selectively localized to the acrosomes of mammalian sperm

    PubMed Central

    1995-01-01

    Calcium flux is required for the mammalian sperm acrosome reaction, an exocytotic event triggered by egg binding, which results in a dramatic rise in sperm intracellular calcium. Calcium-dependent membrane fusion results in the release of enzymes that facilitate sperm penetration through the zona pellucida during fertilization. We have characterized inositol 1,4,5-trisphosphate (IP3)-gated calcium channels and upstream components of the phosphoinositide signaling system in mammalian sperm. Peptide antibodies colocalized G alpha q/11 and the beta 1 isoform of phospholipase C (PLC beta 1) to the anterior acrosomal region of mouse sperm. Western blotting using a polyclonal antibody directed against purified brain IP3 receptor (IP3R) identified a specific 260 kD band in 1% Triton X-100 extracts of rat, hamster, mouse and dog sperm. In each species, IP3R immunostaining localized to the acrosome cap. Scatchard analysis of [3H]IP3 binding to rat sperm sonicates revealed a curvilinear plot with high affinity (Kd = 26 nM, Bmax = 30 pmol/mg) and low affinity (Kd = 1.6 microM, Bmax = 550 pmol/mg) binding sites, reflecting among the highest receptor densities in mammalian tissue. Immunoelectron microscopy confirmed the acrosomal localization in rat sperm. The IP3R fractionated with acrosomes by discontinuous sucrose gradient centrifugation and was enriched in the medium of acrosome- reacted sperm. ATP-dependent 45Ca2+ loading of digitonin permeabilized rat sperm was decreased by 45% in the presence of 10 microM IP3. The IP3-mediated release of calcium was blocked by heparin. Thapsigargin, a sequiterpene lactone inhibitor of the microsomal Ca(2+)-ATPase, stimulated the acrosome reaction of mouse sperm to the same extent as the Ca2+ ionophore, A23187. The failure of caffeine and ryanodine to affect calcium accumulation suggested that thapsigargin acted through an IP3-sensitive store. The presence of G alpha q/11, PLC beta 1 and a functional IP3R in the anterior acrosomal region

  10. Transitional States of Acrosomal Exocytosis and Proteolytic Processing of the Acrosomal Matrix in Guinea Pig Sperm

    PubMed Central

    Kim, Kye-Seong; Foster, James A.; Kvasnicka, Kevin W.; Gerton, George L.

    2011-01-01

    In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the zona pellucida (by what visually appear to be “acrosome-intact” sperm), maintenance of zona pellucida binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the zona pellucida. We feel this “transitional states” model provides a more refined view of acrosomal function that supports a move away from the widely-held, overly simplistic, and binary “acrosome-reaction” model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in zona pellucida binding and penetration. PMID:21919109

  11. Transitional states of acrosomal exocytosis and proteolytic processing of the acrosomal matrix in guinea pig sperm.

    PubMed

    Kim, Kye-Seong; Foster, James A; Kvasnicka, Kevin W; Gerton, George L

    2011-12-01

    In this study, we adapted a FluoSphere bead-binding assay to study the exposure and release of guinea pig sperm acrosomal components during the course of capacitation and acrosomal exocytosis. Prior to capacitation or the initiation of exocytosis, acrosomal proteins were not accessible to FluoSpheres coated with antibodies against two acrosomal matrix (AM) proteins, AM67 and AM50; during the course of capacitation and ionophore-induced acrosomal exocytosis, however, we detected the transient exposure of the solid-phase AM proteins on the surface of guinea pig sperm using the antibody-coated fluorescent beads. Several different transitional stages leading to complete acrosomal exocytosis were classified, and we propose these represent true, functional intermediates since some of the AM proteins are orthologues of mouse proteins that bind the zona pellucida (ZP) of unfertilized eggs. In addition, we present evidence that implicates acrosin in the proteolytic processing of AM50 during AM disassembly. Thus, we propose that the transitional states of acrosomal exocytosis involve early binding of AM proteins to the ZP (by what visually appear to be "acrosome-intact" sperm), maintenance of ZP binding that coincides with the progressive exposure of AM proteins, and gradual proteolytic disassembly of the AM to allow sperm movement through the ZP. We feel this "transitional states" model provides a more refined view of acrosomal function that supports a move away from the widely held, overly simplistic, and binary "acrosome-reaction" model, and embraces a more dynamic view of acrosomal exocytosis that involves intermediate stages of the secretory process in ZP binding and penetration.

  12. Changes in the distribution and molecular mass of boar sperm acrosome-associated 1 proteins during the acrosome reaction; their validity as indicators for occurrence of the true acrosome reaction.

    PubMed

    Ogura, Yukari; Takagishi, Yuki; Harayama, Hiroshi

    2016-09-01

    The aims of this study were to investigate changes in the distribution and molecular mass of boar sperm acrosome-associated 1 (SPACA1) proteins during the acrosome reaction and to discuss validity of SPACA1 proteins as indicators for occurrence of the true acrosome reaction. Boar ejaculated spermatozoa were used for induction of the extracellular Ca(2+)-dependent acrosome reaction (true acrosome reaction) or acrosomal damages (false acrosome reaction) and then subjected to double staining with the anti-SPACA1 protein antibody and FITC-PNA and Western blotting. Extracellular Ca(2+)-dependently acrosome-reacted spermatozoa were characterized by appearance of SPACA1 proteins in the postacrosomal region (; these spermatozoa were classified into SP-3&AR pattern of double staining). However, SPACA1 proteins were not observed in the postacrosomal region of frozen-thawed spermatozoa with severely damaged acrosomes (; these spermatozoa were classified into SP-2&AR pattern). Moreover, the spermatozoa in which acrosomes were severely damaged by incubation with cyclodextrins and without CaCl2 were classified into either SP-2&AR or SP-3&AR pattern. Although SPACA1 proteins were detected mainly as 36-42kDa proteins in the spermatozoa with intact acrosomes, small types of SPACA1 proteins (15-28kDa) increased in extracellular Ca(2+)-dependently acrosome-reacted spermatozoa as well as frozen-thawed spermatozoa with damaged acrosomes. These results show the increase of boar spermatozoa classified into SP-3&AR pattern after incubation in the medium with CaCl2 and without cyclodextrins indicates occurrence of the true acrosome reaction. Moreover, we suggest the increase of small types of SPACA1 proteins is a valid indicator for occurrence of the acrosomal disintegration arising from the true and false acrosome reactions.

  13. [Impact of sperm DNA and acrosome integrity and acrosome reaction rate on outcomes of rescue intracytoplasmic sperm injection].

    PubMed

    He, Yongzhi; Li, Dawen; Cheng, Junping; Huo, Zhongchao; Huang, Hongyi; Xiao, Xin

    2016-01-01

    Objective To explore the effects of sperm DNA integrity rate, acrosome integrity rate and acrosome reaction rate on the outcomes of rescue intracytoplasmic sperm injection (ICSI). This retrospective analysis was conducted among 97 infertile couples receiving rescue ICSI due to failure of in vitro fertilization procedures in our Reproductive Medicine Center. Of these 97 women, 41 had clinical pregnancy and 56 did not, and the effects of sperm DNA integrity rate (estimated by DNA fragmentation index, DFI), acrosome integrity rate and acrosome reaction rate on rescue ICSI outcomes were analyzed. No significant difference was found in paternal age, testosterone value, testicular volume, FSH, female patient' age or the number of eggs retrieved between the two groups (P>0.05), but the infertility years was significantly shorter in the pregnancy group than in the non-pregnancy group (P<0.05). The fertilization rate and cleavage rate were similar between the two groups (P>0.05), but the good embryo rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity or acrosome reaction rate did not differ significantly between the two groups (P>0.05), but the acrosome integrity rate was significantly higher in the pregnancy group (P<0.05). The sperm DNA integrity rate, acrosome integrity or acrosome reaction rate were not correlated with the fertilization rate, cleavage rate or good embryo rate (P>0.05). The pregnancy rate, twin and single fetus rates were 42.3%, 10.3% and 32.0% in this cohort after recue ICSI, respectively. Rescue ICSI is an effective treatment after failed in vitro fertilization procedure, and sperm acrosome integrity rate is associated with the outcome of rescue ICSI.

  14. Isolation and Proteomic Characterization of the Mouse Sperm Acrosomal Matrix*

    PubMed Central

    Guyonnet, Benoit; Zabet-Moghaddam, Masoud; SanFrancisco, Susan; Cornwall, Gail A.

    2012-01-01

    A critical step during fertilization is the sperm acrosome reaction in which the acrosome releases its contents allowing the spermatozoa to penetrate the egg investments. The sperm acrosomal contents are composed of both soluble material and an insoluble material called the acrosomal matrix (AM). The AM is thought to provide a stable structure from which associated proteins are differentially released during fertilization. Because of its important role during fertilization, efforts have been put toward isolating the AM for biochemical study and to date AM have been isolated from hamster, guinea pig, and bull spermatozoa. However, attempts to isolate AM from mouse spermatozoa, the species in which fertilization is well-studied, have been unsuccessful possibly because of the small size of the mouse sperm acrosome and/or its fusiform shape. Herein we describe a procedure for the isolation of the AM from caput and cauda mouse epididymal spermatozoa. We further carried out a proteomic analysis of the isolated AM from both sperm populations and identified 501 new proteins previously not detected by proteomics in mouse spermatozoa. A comparison of the AM proteome from caput and cauda spermatozoa showed that the AM undergoes maturational changes during epididymal transit similar to other sperm domains. Together, our studies suggest the AM to be a dynamic and functional structure carrying out a variety of biological processes as implied by the presence of a diverse group of proteins including proteases, chaperones, hydrolases, transporters, enzyme modulators, transferases, cytoskeletal proteins, and others. PMID:22707618

  15. Pentoxifylline increases sperm penetration into zona-free hamster oocytes without increasing the acrosome reaction.

    PubMed

    Morales, P; Llanos, M; Yovich, J L; Cummins, J M; Vigil, P

    1993-01-01

    Several drugs have been used to stimulate human sperm motility, including 3-deoxy-adenosine, caffeine, and pentoxifylline. Pentoxifylline is an inhibitor of the phosphodiesterase and may stimulate sperm motility by increasing the intracellular levels of cAMP. In this study we have evaluated the effect of pentoxifylline in the outcome of the sperm penetration assay into zona-free hamster oocytes. Twenty-seven semen samples, obtained for diagnostic purposes, were used. After the motile sperm were selected by the swim-up technique, the samples were divided into two aliquots. One aliquot was incubated with 1 mg ml-1 of pentoxifylline at 37 degrees C, 5% CO2 for 30 min. The control aliquot was incubated with culture medium. The samples were then washed and resuspended in fresh, pentoxifylline-free medium, at a sperm concentration of 10 x 10(6) cells ml-1. One hundred microlitres of each sperm suspension was then deposited under oil and 30-40 zona-free hamster oocytes were added. After 6 h of gamete coincubation, the percentage of penetrated oocytes and the number of decondensed sperm heads were evaluated. The percentage of acrosome-reacted sperm was evaluated using the Pisum sativum lectin. The percentage of zona-free hamster oocytes penetrated was increased after pentoxifylline-treatment. The percentage of acrosome reacted sperm and the number of decondensed sperm heads per egg were not different between the control and the pentoxifylline-treated groups. The results suggest that the beneficial effect of pentoxifylline upon the sperm cells is not mediated by stimulation of the acrosome reaction.

  16. Elucidation of the Involvement of p14, a Sperm Protein during Maturation, Capacitation and Acrosome Reaction of Caprine Spermatozoa

    PubMed Central

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C.

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event. PMID:22291985

  17. Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

    PubMed

    Nandi, Pinki; Ghosh, Swatilekha; Jana, Kuladip; Sen, Parimal C

    2012-01-01

    Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

  18. Molecular basis of sperm capacitation acrosome reaction and interaction with eggs

    SciTech Connect

    Sheikhnejad, G.

    1985-01-01

    A phospholipase C (PLC) which can hydrolyze /sup 14/C-phosphatidylcholine was purified from bull seminal plasma. This PLC has an optimum at pH 7.2 and its PI was about 5.0. The enzyme was inhibited by EDTA, Cd/sup 2 +/, Pb/sup 2 +/, Ni/sup 2 +/, Fe/sup 2 +/, and Zn/sup 2 +/. PLC consists of two subunits one 69,000 and the other 55,000 daltons. The purified PLC was examined for induction of capacitation and acrosome reaction of guinea pig spermatozoa. Sperm were examined for the acrosome reaction 10 min after addition of 3.4 mM Ca/sup 2 +/. Fifty percent of the sperm underwent the acrosome reaction while the control had less than 5% acrosome reacted sperm. The antiserum to the inneracrosomal membrane isolated from sperm was labeled with FITC conjugated goat anti-guinea pig IgG. The conjugated antibody was used to localize sperm antigens. The antigens located on the IAM were only fluoresced when rabbit sperm were treated with methanol and/or MgCl/sub 2/. Therefore anti-IAM antibody did not bind to the sperm plasma membrane. In vivo capacitated rabbit sperm were incubated with anti-IAM antibody (intact IgG and F(ab')/sub 2/ fragments) for 30 min prior to addition of rabbit eggs. After 24 h the eggs were examined for cleavage. The control eggs were fertilized (90%) while the antibody completely inhibited the fertilization of ova in vitro. The eggs incubated with antibody prior to the addition of sperm were still fertilizable. Thus, anti-IAM did not have any noticeable effect on the eggs. It was also shown that antibody inhibited fertilization of zona-free rabbit eggs in vitro as well.

  19. Expanded cumuli induce acrosome reaction in boar sperm.

    PubMed

    Mattioli, M; Lucidi, P; Barboni, B

    1998-12-01

    The authors investigated acrosomal changes occurring in boar sperm that interact with the expanded cumulus matrix surrounding ovulated pig oocytes. Samples of washed boar sperm obtained from six donors were incubated for 4 hr under capacitating conditions and exposed either to solubilized zonae pellucidae (ZP) or solubilized expanded pig cumuli (SEC) obtained from IVM oocytes. Alternatively, hyaluronic acid, laminin, or fibronectin, components of the extracellular matrix (ECM) were added to capacitated sperm. Acrosomal integrity was evaluated 1 hr later by using FITC-PSA staining. Solubilized cumuli induced acrosome reaction (AR) in a dose-dependent manner with a saturating effect exerted at 2.5 SEC/50 microl. Both 500 nM fibronectin and 500 nM laminin stimulated acrosomal exocytosis, the latter being more effective and inducing saturating levels of AR. By contrast, hyaluronic acid did not affect acrosomal status. Preincubation with anti-laminin antibodies completely prevented the inducing activity of SEC without affecting the activity of solubilized ZP. Consistent with these data, the integrin VLA-6, a receptor with high affinity for laminin, was detected by immunoblotting on the plasma membrane of capacitated boar spermatozoa. In addition, its immunoneutralization, obtained with the preincubation of capacitated sperm with the antibody raised against the alpha chain of VLA-6 integrin, prevented AR upon exposure to laminin or SEC (10.7+/-3.2 and 10.2+/-1.0% respectively), while the samples retained their responsiveness to ZP (29.6+/-1.2%). The results demonstrate that the interaction between laminin, entrapped in the expanded cumuli, and specific integrins present on the sperm membrane can initiate AR, thus taking part in the process of sperm-egg recognition.

  20. Modern human sperm freezing: Effect on DNA, chromatin and acrosome integrity.

    PubMed

    Rahiminia, Tahereh; Hosseini, Akram; Anvari, Morteza; Ghasemi-Esmailabad, Saeed; Talebi, Ali Reza

    2017-08-01

    Presence of vitrification method in sperm freezing and the introduction of solid surface vitrification beside rapid freezing in vapour, opens an easy and safe way to help infertility centres. While the effects of cryopreservation on motility, morphology and viability of sperm are documented, the question of the probable alteration of sperm DNA, chromatin and acrosome integrity after freezing and thawing procedures in different methods is still controversial. Normal sample were collected according to WHO strict criteria. Sperm suspensions were mixed 1:1 with 0.5 M sucrose and divided into four equal aliquots for freezing: fresh, nitrogen direct immersion vitrification (Vit), solid surface vitrification (SSV) and in vapour (Vapour). Sperm suspensions were transferred into a 0.25 ml sterile plastic. Then straw was inserted inside the 0.5 ml straw. For thawing, the straws were immersed in a 42 °C water bath. Beside the sperm parameters, we assessed the acrosome reaction by double staining, chromatin integrity by toluidine blue (Tb) and chromomycin A3 (CMA3) and DNA integrity by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) respectively. In progressive motility, the highest rate occurred in Vit (39.9 ± 13.3). Moreover, the lowest rate of immotile sperm was in Vit (32.7 ± 16.3). In normal morphology, the group Vit was similar to the fresh, while SSV and Vapour were significantly different from the fresh. The percentage of acrosome-reacted sperms was more in Vit (81.3 ± 10.2) than the fresh group. TUNEL+ results showed that DNA fragmentation was significantly increased in Vit (p-value = 0.025). While in SSV and Vapour results were comparable to fresh. There was a significant correlation between TUNEL+ and normal morphology, TB, CMA3 and presence of intact acrosome. Sperm in Vapour was healthier in terms of DNA, chromatin and acrosome integrity. In contrast of higher motility and normal morphology; DNA, chromatin and acrosome

  1. Effect of cholesterol-loaded-cyclodextrin on sperm viability and acrosome reaction in boar semen cryopreservation.

    PubMed

    Lee, Yong-Seung; Lee, Seunghyung; Lee, Sang-Hee; Yang, Boo-Keun; Park, Choon-Keun

    2015-08-01

    This study was undertaken to examine the effect of cholesterol-loaded-cyclodextrin (CLC) on boar sperm viability and spermatozoa cryosurvival during boar semen cryopreservation, and methyl-β-cyclodextrin (MBCD) was treated for comparing with CLC. Boar semen treated with CLC and MBCD before freezing process to monitor the effect on survival and capacitation status by flow cytometry with appropriate fluorescent probes. Sperm viability was higher in 1.5mg CLC-treated sperm (76.9±1.01%, P<0.05) than un-treated and MBCD-treated sperm before cryopreservation (58.7±1.31% and 60.3±0.31%, respectively). For CTC patterns, F-pattern was higher in CLC treated sperm than MBCD-treated sperm, for B-pattern was higher in CLC-treated sperm than fresh sperm (P<0.05). For AR pattern (an acrosome-reacted sperm) was lower in CLC-treated sperm than MBCD-treated sperm (P<0.05). Moreover, we examined in vitro development of porcine oocytes after in vitro fertilization using CLC-treated frozen-thawed semen, in which CLC treatment prior to freezing and thawing increased the development of oocytes to blastocyst stage in vitro. In conclusion, CLC could protect the viability of spermatozoa from cryodamage prior to cryopreservation in boar semen.

  2. Chronic restraint stress induces sperm acrosome reaction and changes in testicular tyrosine phosphorylated proteins in rats

    PubMed Central

    Arun, Supatcharee; Burawat, Jaturon; Sukhorum, Wannisa; Sampannang, Apichakan; Maneenin, Chanwit; Iamsaard, Sitthichai

    2016-01-01

    Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein. PMID:27525328

  3. Using quantitative interference phase microscopy for sperm acrosome evaluation (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Balberg, Michal; Kalinowski, Ksawery; Levi, Mattan; Shaked, Natan T.

    2016-03-01

    We demonstrate quantitative assessment of sperm cell morphology, primarily acrosomal volume, using quantitative interference phase microscopy (IPM). Normally, the area of the acrosome is assessed using dyes that stain the acrosomal part of the cell. We have imaged fixed individual sperm cells using IPM. Following, the sample was stained and the same cells were imaged using bright field microscopy (BFM). We identified the acrosome using the stained BFM image, and used it to define a quantitative corresponding area in the IPM image and determine a quantitative threshold for evaluating the volume of the acrosome.

  4. Con A-binding protein Zn-α2-glycoprotein on human sperm membrane is related to acrosome reaction and sperm fertility.

    PubMed

    Liu, Y; Qu, F; Cao, X; Chen, G; Guo, Q; Ying, X; Guo, W; Lu, L; Ding, Z

    2012-04-01

    Fertilization, the recognition and fusion between spermatozoa and oocyte, involves various molecules on the spermatozoa and oocyte membranes. Concanavalin A (ConA)-binding proteins may be one of the molecules involved in mammal spermatozoa fertilization; however, their structure and function remain largely unknown. Here, we initially identified a ConA-binding protein, Zn-α2-glycoprotein (ZAG), involved in regulating the acrosome reaction (AR) of human spermatozoa. ZAG is localized on the pre-equatorial region covering the acrosome, neck and tail (some parts of middle piece and principal piece respectively) regions of the acrosome intact human spermatozoa, and disappears in the acrosomal region of the acrosome-reacted spermatozoa. Polyclonal antibodies against human recombinant ZAG significantly reduced the AR and sperm capability binding to human zona pellucida or penetration into zona-free hamster oocytes. Furthermore, assessment of the signaling pathways regulated by ZAG revealed that ZAG affects sperm AR through both the cAMP/PKA and PKC pathways. These results indicate that ZAG, which is present on the human sperm membrane, plays a critical role in the AR and subsequently, may be involved in sperm fertility.

  5. The laminin-induced acrosome reaction in human sperm is mediated by Src kinases and the proteasome.

    PubMed

    Tapia, Silvia; Rojas, Marcelo; Morales, Patricio; Ramirez, Marco A; Diaz, Emilce S

    2011-08-01

    The aim of this work was to determine whether laminin (Ln), an extracellular matrix protein, induces the intracellular events that may be involved in producing the acrosome reaction in human sperm. To this end, we evaluated the effect of Ln on tyrosine phosphorylation, intracellular calcium concentration, proteasome activity, and phosphorylation in human sperm. Aliquots of highly motile sperm selected with a Percoll gradient, were incubated with different concentrations of Ln (0-20 μg/ml) for different periods (0-18 h). The percentage of viable acrosome-reacted sperm was evaluated using fluorescein isothiocyanate-labeled Pisum sativum agglutinin and Hoechst 33258 DNA dye. Tyrosine phosphorylation was evaluated by Western blot analysis. The chymotrypsin-like activity of the proteasome was evaluated with a fluorogenic peptide, and intracellular calcium concentration was measured with fura-2. The results indicate that Ln stimulated the acrosome reaction of human sperm in a dose-dependent manner. This increase was drastically inhibited in the presence of herbimycin A, SU6656, and epoxomicin. In addition, Ln increased proteasome activity and phosphorylation; both events were inhibited by herbimycin A and SU6656. Finally, Ln induced an increase in intracellular calcium concentration, which was inhibited by SU6656 and epoxomicin. These results suggest that Ln is able to induce the acrosome reaction. This effect may be mediated by Src kinase and the proteasome, with the consequent induction of a calcium influx.

  6. Identification of Bovine Sperm Acrosomal Proteins that Interact with a 32kDa Acrosomal Matrix Protein

    PubMed Central

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-01-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction is comprised of three major (54, 50, and 45kDa) and several minor (38–19kDa) polypeptides. The set of minor polypeptides (38–19kDa) termed “OMCrpf polypeptides” is selectively solubilized by high-pH extraction (pH 10.5) while the three major polypeptides (55, 50 and 45kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38–19kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment; whereas, IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidyl choline (LPC

  7. Identification of bovine sperm acrosomal proteins that interact with a 32-kDa acrosomal matrix protein.

    PubMed

    Nagdas, Subir K; Smith, Linda; Medina-Ortiz, Ilza; Hernandez-Encarnacion, Luisa; Raychoudhury, Samir

    2016-03-01

    Mammalian fertilization is accomplished by the interaction between sperm and egg. Previous studies from this laboratory have identified a stable acrosomal matrix assembly from the bovine sperm acrosome termed the outer acrosomal membrane-matrix complex (OMC). This stable matrix assembly exhibits precise binding activity for acrosin and N-acetylglucosaminidase. A highly purified OMC fraction comprises three major (54, 50, and 45 kDa) and several minor (38-19 kDa) polypeptides. The set of minor polypeptides (38-19 kDa) termed "OMCrpf polypeptides" is selectively solubilized by high-pH extraction (pH 10.5), while the three major polypeptides (55, 50, and 45 kDa) remain insoluble. Proteomic identification of the OMC32 polypeptide (32 kDa polypeptide isolated from high-pH soluble fraction of OMC) yielded two peptides that matched the NCBI database sequence of acrosin-binding protein. Anti-OMC32 recognized an antigenically related family of polypeptides (OMCrpf polypeptides) in the 38-19-kDa range with isoelectric points ranging between 4.0 and 5.1. Other than glycohydrolases, OMC32 may also be complexed to other acrosomal proteins. The present study was undertaken to identify and localize the OMC32 binding polypeptides and to elucidate the potential role of the acrosomal protein complex in sperm function. OMC32 affinity chromatography of a detergent-soluble fraction of bovine cauda sperm acrosome followed by mass spectrometry-based identification of bound proteins identified acrosin, lactadherin, SPACA3, and IZUMO1. Co-immunoprecipitation analysis also demonstrated the interaction of OMC32 with acrosin, lactadherin, SPACA3, and IZUMO1. Our immunofluorescence studies revealed the presence of SPACA3 and lactadherin over the apical segment, whereas IZUMO1 is localized over the equatorial segment of Triton X-100 permeabilized cauda sperm. Immunoblot analysis showed that a significant portion of SPACA3 was released after the lysophosphatidylcholine (LPC)-induced acrosome

  8. The osmotic properties of the acrosome of guinea-pig sperm.

    PubMed

    Green, D P

    1978-08-01

    The osmotic behaviour of the acrosome in intact guinea-pig sperm has been examined by light and electron microscopy. Because the acrosome is retained within the cell, it can only experience the changes in tonicity of the cytoplasm and these have first to be related to the changes in tonicity in the external medium. Nevertheless, a qualitative description of the osmotic properties of the acrosome can be obtained. The evidence suggests that the acrosomal contents are normally close to their limit of compression. It has been proposed that a hydrostatic pressure is exerted inwards on the acrosomal membrane and that this is responsible for the maintenance of acrosomal shape. Swelling of the acrosome when sperm are suspended in hypotonic medium suggests that the acrosomal membrane does not have an abnormally low water permeability. Two-thirds of the acrosomal volume are occupied by a single matrix which undergoes cavitation in the course of the acrosome reaction. It also undergoes cavitation when guinea-pig sperm are suspended in calcium-free medium containing the non-ionic detergent Triton X-100. This suggests that cavitation is caused by the loss of integrity of the plasma and acrosomal membranes. A mechanism for cavitation is proposed in which a colloid osmotic pressure within the acrosomal matrix is allowed expression when the crystalloids of the external medium pass into it, this internal colloid osmotic pressure forcing the matrix apart to produce a cavity.

  9. In vitro capacitation and acrosome reaction in sperm of the phyllostomid bat Artibeus jamaicensis.

    PubMed

    Álvarez-Guerrero, Alma; González-Díaz, Francisco; Medrano, Alfredo; Moreno-Mendoza, Norma

    2016-04-01

    Sperm capacitation occurs during the passage of sperm through the female reproductive tract. Once the sperm binds to the pellucid zone, the acrosome reaction to enable penetration of the oocyte is completed. In this study, sperm of Artibeus jamaicensis bat was used to evaluate both capacitation status and the acrosome reaction under in vitro conditions, incubating sperm at 32 and 37°C with and without progesterone. Sperm was incubated at different times to assess sperm cells' functionality in terms of capacitation and acrosome reaction, using the chlortetracycline staining, lectin fluoresceinisocyanate conjugate-Pisum sativum agglutinin (FITC-PSA), and transmission electron microscopy. Sperm cells that presented uniform fluorescence throughout the head and mid-piece were classified as non-capacitated. Subsequently, sperm cells, which were observed with fluorescence only in the anterior portion of the head and mid-piece, were classified as capacitated. Sperm cells with no fluorescence in the head, but fluorescence in the mid-piece, were categorized as sperm cells that have carried out the acrosome reaction. During the acrosome reaction, sperm cells showed changes in their morphology, so it was not possible to distinguish the plasma and acrosomal membranes. Around the entire head, it was not possible to distinguish the fusion points between these membranes that made it possible for the acrosomal reaction to take place and thus to release the enzymes necessary to penetrate the pellucid zone. In conclusion, under appropriate in vitro conditions and by supplementing the culture medium with progesterone, A. jamaicensis bat sperm cells are able to be capacitated in a period from 6 to 8 h and to carry out the acrosome reaction.

  10. Mouse Sperm Membrane Potential Hyperpolarization Is Necessary and Sufficient to Prepare Sperm for the Acrosome Reaction*

    PubMed Central

    De La Vega-Beltran, Jose Luis; Sánchez-Cárdenas, Claudia; Krapf, Darío; Hernandez-González, Enrique O.; Wertheimer, Eva; Treviño, Claudia L.; Visconti, Pablo E.; Darszon, Alberto

    2012-01-01

    Mammalian sperm are unable to fertilize the egg immediately after ejaculation; they acquire this capacity during migration in the female reproductive tract. This maturational process is called capacitation and in mouse sperm it involves a plasma membrane reorganization, extensive changes in the state of protein phosphorylation, increases in intracellular pH (pHi) and Ca2+ ([Ca2+]i), and the appearance of hyperactivated motility. In addition, mouse sperm capacitation is associated with the hyperpolarization of the cell membrane potential. However, the functional role of this process is not known. In this work, to dissect the role of this membrane potential change, hyperpolarization was induced in noncapacitated sperm using either the ENaC inhibitor amiloride, the CFTR agonist genistein or the K+ ionophore valinomycin. In this experimental setting, other capacitation-associated processes such as activation of a cAMP-dependent pathway and the consequent increase in protein tyrosine phosphorylation were not observed. However, hyperpolarization was sufficient to prepare sperm for the acrosome reaction induced either by depolarization with high K+ or by addition of solubilized zona pellucida (sZP). Moreover, K+ and sZP were also able to increase [Ca2+]i in non-capacitated sperm treated with these hyperpolarizing agents but not in untreated cells. On the other hand, in conditions that support capacitation-associated processes blocking hyperpolarization by adding valinomycin and increasing K+ concentrations inhibited the agonist-induced acrosome reaction as well as the increase in [Ca2+]i. Altogether, these results suggest that sperm hyperpolarization by itself is key to enabling mice sperm to undergo the acrosome reaction. PMID:23095755

  11. Acrosomal reaction of thyone sperm. I. Changes in the sperm head visualized by high resolution video microscopy

    PubMed Central

    1982-01-01

    Structural changes inside the head of Thyone sperm undergoing the acrosomal reaction were followed with a high-resolution, differential interference contrast (DIC) video microscope. The beating sperm, adhering by their midpiece to the cover slip of a wedge perfusion chamber, were activated by a calcium ionophore (20 microM A23187) suspended in sea water containing 50 mM excess CaCl2. Before activation of the sperm, the acrosomal region appears as a 1.1-microM diameter sphere, slightly less dense than the rest of the sperm head. Upon activation, the acrosome pops; the acrosomal region suddenly swells and its refractive index drops. After approximately 1 s, a crescent-shaped periacrosomal cup appears behind the acrosomal vacuole. In the next several seconds, the cup loses more refractive index and expands forward as the acrosomal process extends. The acrosomal vacuole becomes smaller, but without appreciable drop in refractive index. These observations, coupled with the behavior of the extending acrosomal process reported in the companion paper, and in electron microscopy (EM) and early physiological studies, suggest that the acrosomal process is extended by a combination of the explosive polymerization of actin and the osmotic swelling of the periacrosomal cup material. In this paper, we also consider the meaning of the enhanced DIC image seen in the high-resolution video microscope, and discuss the reliability of measurements on small linear dimensions made with the DIC microscope. PMID:6811599

  12. [Detection of human sperm morphology and acrosome reaction with Coomassie brilliant blue staining].

    PubMed

    Lu, Hai-Yi; Lu, Jin-Chun; Hu, Yu-An; Wang, Yong-Mei; Huang, Yu-Feng

    2002-01-01

    To evaluate the application of Coomassie brilliant blue (CBB) G250 staining for the detection of human sperm deformity rate, rate of intact acrosome and acrosome reaction. The smear of spermatozoa before and after capacitation and induced acrosome reaction with progesterone (P) were stained with 0.05% CBB G250 and Wright-Giemisa solution respectively, and visualized with light microscopy. The deformity rate of spermatozoa, rate of intact acrosome and acrosome reaction were calculated. There was no any difference in detection of deformity rate of spermatozoa and rate of intact acrosome with CBB G250 and Wright-Giemisa staining(P < 0.05). The sperm population of acrosome reaction with induced P was divided by CBB staining into two types: positive staining with dark violet blue on acrosome cap and pale or negative staining on the same area. The rate of the latter was increasing with increasing inductive time, maybe representative of the rate of acrosome reaction. The mean rate was(75.1 +/- 3.8)% after induced for 1 h. CBB G250 staining is a reliable method for assessment of the human sperm morphology and acrosome reaction.

  13. Acrosome-reacted mouse spermatozoa recovered from the perivitelline space can fertilize other eggs

    PubMed Central

    Inoue, Naokazu; Satouh, Yuhkoh; Ikawa, Masahito; Okabe, Masaru; Yanagimachi, Ryuzo

    2011-01-01

    Many investigators maintain that spermatozoa that have initiated the acrosome reaction (AR) before reaching the surface of the egg's zona pellucida (ZP) are unable to bind and penetrate the ZP. A recent study has revealed that most fertilizing mouse spermatozoa initiate the AR before contacting the ZP. We found that acrosome-reacted spermatozoa collected from the perivitelline space of Cd9-null mice (whose egg plasma membranes are incapable of fusing with spermatozoa) were able to pass through both the cumulus and ZP of WT mouse eggs and produced live offspring. This means that the spermatozoa we used had the ability to pass through the ZP at least twice. Apparently, some spermatozoa that had undergone the AR long before contact with the ZP remained capable of crossing the ZP and fertilizing eggs. Thus, the concept that acrosome-reacted spermatozoa are unable to bind to the ZP and have lost their fertilizing capacity must be reconsidered. PMID:22084105

  14. Effect of Akti-2 on sperm motility, capacitation and acrosome reaction in a mouse model.

    PubMed

    Quan, Yanmei; Liu, Qiang

    2016-05-01

    The aim of the present study was to investigate the effect of the Akt inhibitor, Akti-2, on the sperm motility and acrosome reaction in mice. Mature sperms from the adult mice, aged 8 weeks, were co-incubated with Akti-2 for ~30 min at 37°C in 5% CO2, and the sperm viability was assessed by eosin-nigrosin staining. The sperm total and progressive motility were analyzed by computer-aided sperm analysis. In addition, the acrosome reaction of sperms was detected by the acid phosphatase assay, Coomassie Brilliant Blue staining and fluorescein-isothiocyanate conjugated pisum sativum lectin staining, respectively. Compared with the control (dimethyl sulfoxide), Akti-2 had no effect on sperm viability, but it suppressed the total and progressive motility significantly. Furthermore, the capacitation-associated protein tyrosine phosphorylation and the acrosome reaction induced by calcium ionophore A23187 could be suppressed by Akti-2. These experiments confirmed that Akti-2 significantly impaired the sperm functions, including motility, capacitation and acrosome reaction, and provide the proof for its potential in male reproductive toxicity.

  15. Effect of Akti-2 on sperm motility, capacitation and acrosome reaction in a mouse model

    PubMed Central

    QUAN, YANMEI; LIU, QIANG

    2016-01-01

    The aim of the present study was to investigate the effect of the Akt inhibitor, Akti-2, on the sperm motility and acrosome reaction in mice. Mature sperms from the adult mice, aged 8 weeks, were co-incubated with Akti-2 for ~30 min at 37°C in 5% CO2, and the sperm viability was assessed by eosin-nigrosin staining. The sperm total and progressive motility were analyzed by computer-aided sperm analysis. In addition, the acrosome reaction of sperms was detected by the acid phosphatase assay, Coomassie Brilliant Blue staining and fluorescein-isothiocyanate conjugated pisum sativum lectin staining, respectively. Compared with the control (dimethyl sulfoxide), Akti-2 had no effect on sperm viability, but it suppressed the total and progressive motility significantly. Furthermore, the capacitation-associated protein tyrosine phosphorylation and the acrosome reaction induced by calcium ionophore A23187 could be suppressed by Akti-2. These experiments confirmed that Akti-2 significantly impaired the sperm functions, including motility, capacitation and acrosome reaction, and provide the proof for its potential in male reproductive toxicity. PMID:27123250

  16. Identification and Characterization of a Bovine Sperm Acrosomal Matrix Protein and its Mechanism of Interaction with Acrosomal Hydrolases

    PubMed Central

    Nagdas, Subir K; Smith, Linda; Mcnamara, Allen; Hernandez–Encarnacion, Luisa; Medina-Ortiz, Ilza

    2015-01-01

    Fertilization, the union of male and female gametes to create offspring, is an intricate biological process dependent upon several biochemical and physiological events. Our understanding of the functions of protein constituents of the outer acrosomal membrane-associated matrix complex (OMC) is limited. A highly purified OMC fraction isolated from bovine cauda sperm heads is comprised of 54, 50, 45, and 38–19kDa polypeptides. The objective of this study is to identify and to characterize the 45kDa (OMC45) polypeptide and to define its role in binding acrosomal hydrolases and to examine the fate of OMC45 polypeptide during the acrosome reaction. We isolated OMC45 polypeptide from the high-pH insoluble fraction of OMC. Proteomic analysis of OMC45 by MALDI–TOF–TOF yielded 8 peptides that matched the NCBI database sequence of Tektin 3 (TEKT3). Triton X–100–permeabilized cauda sperm exhibited intense staining of the acrosomal segment with anti–OMC45 and anti–TEKT3. The OMC45 polypeptide was solubilized by RIPA (radio-immunoprecipitation assay) buffer extraction. The solubilized fraction was subjected to immunoprecipitation analysis. The OMC45 polypeptide was recovered in the anti–OMC45 immunoprecipitation pellet. An identical blot stained with anti–TEKT3 exhibited the presence of TEKT3 polypeptide in the anti–OMC45 pellet. Our immunofluorescence and biochemical studies confirm the proteomics identification of OMC45 polypeptide; that it exhibits a sequence similarity to TEKT3. OMC45 glycoprotein possesses both N–linked and O–linked oligosaccharides. Deglycosylated OMC45 revealed a significant reduction in both acrosin and N–acetylglucosaminidase (NAGA) binding in comparison with acrosin and NAGA binding to a native OMC45 polypeptide, demonstrating the important role of oligosaccharides in hydrolase binding. OMC45 polypeptide is not released during the acrosome reaction but remains in the particulate cell subfraction, associated with the hybrid

  17. Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm

    SciTech Connect

    Bleil, J.D.; Wassarman, P.M.

    1986-04-01

    The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only background levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.

  18. ERK1/2 mediates sperm acrosome reaction through elevation of intracellular calcium concentration.

    PubMed

    Jaldety, Yael; Breitbart, Haim

    2015-10-01

    Mammalian sperm acquire fertilization capacity after residing in the female reproductive tract for a few hours in a process called capacitation. Only capacitated sperm can bind the zona pellucida (ZP) of the egg and undergo the acrosome reaction, a process that allows penetration and fertilization. Extracellular signal regulated kinase (ERK1/2) mediates signalling in many cell types, however its role in sperm function is largely unknown. Here we show that ERK1/2 is highly phosphorylated/activated after a short incubation of mouse sperm under capacitation conditions and that this phosphorylation is reduced after longer incubation. Further phosphorylation was observed upon addition of crude extract of egg ZP or epidermal growth factor (EGF). The mitogen-activated ERK-kinase (MEK) inhibitor U0126 abolished ERK1/2 phosphorylation, in vitro fertilization rate and the acrosome reaction induced by ZP or EGF but not by the Ca2+-ionophore A23187. Moreover, inhibition of ERK1/2 along the capacitation process diminished almost completely the sperm's ability to go through the acrosome reaction, while inhibition at the end of capacitation attenuated the acrosome reaction rate by only 45%. The fact that the acrosome reaction, induced by the Ca2+ -ionophore A23187, was not inhibited by U0126 suggests that ERK1/2 mediates the acrosome reaction by activating Ca2+ transport into the cell. Direct determination of intracellular [Ca2+] revealed that Ca2+ influx induced by EGF or ZP was completely blocked by U0126. Thus, it has been established that the increase in ERK1/2 phosphorylation/activation in response to ZP or by activation of the EGF receptor (EGFR) by EGF, is a key event for intracellular Ca2+ elevation and the subsequent occurrence of the acrosome reaction.

  19. Characterization of CD46 and β1 integrin dynamics during sperm acrosome reaction

    PubMed Central

    Frolikova, Michaela; Sebkova, Natasa; Ded, Lukas; Dvorakova-Hortova, Katerina

    2016-01-01

    The acrosome reaction (AR) is a process of membrane fusion and lytic enzyme release, which enables sperm to penetrate the egg surroundings. It is widely recognized that specific sperm proteins form an active network prior to fertilization, and their dynamic relocation is crucial for the sperm-egg fusion. The unique presence of the membrane cofactor protein CD46 in the sperm acrosomal membrane was shown, however, its behaviour and connection with other sperm proteins has not been explored further. Using super resolution microscopy, we demonstrated a dynamic CD46 reorganisation over the sperm head during the AR, and its interaction with transmembrane protein integrins, which was confirmed by proximity ligation assay. Furthermore, we propose their joint involvement in actin network rearrangement. Moreover, CD46 and β1 integrins with subunit α3, but not α6, are localized into the apical acrosome and are expected to be involved in signal transduction pathways directing the acrosome stability and essential protein network rearrangements prior to gamete fusion. PMID:27666019

  20. Anandamide (arachidonylethanolamide), a brain cannabinoid receptor agonist, reduces sperm fertilizing capacity in sea urchins by inhibiting the acrosome reaction.

    PubMed Central

    Schuel, H; Goldstein, E; Mechoulam, R; Zimmerman, A M; Zimmerman, S

    1994-01-01

    Anandamide (arachidonylethanolamide) is an endogenous cannabinoid receptor agonist in mammalian brain. Sea urchin sperm contain a high-affinity cannabinoid receptor similar to the cannabinoid receptor in mammalian brain. (-)-delta 9-Tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. We now report that anandamide produces effects similar to those previously obtained with THC in Strongylocentrotus purpuratus in reducing sperm fertilizing capacity and inhibiting the egg jelly-stimulated acrosome reaction. Arachidonic acid does not inhibit the acrosome reaction under similar conditions. The adverse effects of anandamide on sperm fertilizing capacity and the acrosome reaction are reversible. The receptivity of unfertilized eggs to sperm and sperm motility are not impaired by anandamide. Under conditions where anandamide completely blocks the egg jelly-stimulated acrosome reaction, it does not inhibit the acrosome reaction artificially initiated by ionomycin, which promotes Ca2+ influx, and nigericin, which activates K+ channels in sperm. These findings provide additional evidence that the cannabinoid receptor in sperm plays a role in blocking the acrosome reaction, indicate that anandamide or a related molecule may be the natural ligand for the cannabinoid receptor in sea urchin sperm, and suggest that binding of anandamide to the cannabinoid receptor modulates stimulus-secretion-coupling in sperm by affecting an event prior to ion channel opening. PMID:8052642

  1. Silymarin protects plasma membrane and acrosome integrity in sperm treated with sodium arsenite.

    PubMed

    Eskandari, Farzaneh; Momeni, Hamid Reza

    2016-01-01

    Exposure to arsenic is associated with impairment of male reproductive function by inducing oxidative stress. Silymarin with an antioxidant property scavenges free radicals. The aim of this study was to investigate if silymarin can prevent the adverse effects of sodium arsenite on ram sperm plasma membrane and acrosome integrity. Ram epidydimal spermatozoa were divided into five groups: spermatozoa at 0 hr, spermatozoa at 180 min (control), spermatozoa treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 min, spermatozoa treated with sodium arsenite (10 μM) for 180 min and spermatozoa treated with silymarin (20 μM) for 180 min. Double staining of Hoechst and propidium iodide was performed to evaluate sperm plasma membrane integrity, whereas comassie brilliant blue staining was used to assess acrosome integrity. Plasma membrane (p< 0.001) and acrosome integrity (p< 0.05) of the spermatozoa were significantly reduced in sodium arsenite group compared to the control. In silymarin + sodium arsenite group, silymarin was able to significantly (p< 0.001) ameliorate the adverse effects of sodium arsenite on these sperm parameters compared to sodium arsenite group. The incubation of sperm for 180 min (control group) showed a significant (p< 0.001) decrease in acrosome integrity compared to the spermatozoa at 0 hour. The application of silymarin alone for 180 min could also significantly (p< 0.05) increase sperm acrosome integrity compared to the control. Silymarin as a potent antioxidant could compensate the adverse effects of sodium arsenite on the ram sperm plasma membrane and acrosome integrity.

  2. Sustained High Protein-tyrosine Phosphatase 1B Activity in the Sperm of Obese Males Impairs the Sperm Acrosome Reaction*

    PubMed Central

    Shi, Lei; Zhang, Qipeng; Xu, Binqiang; Jiang, Xiaohong; Dai, Yutian; Zhang, Chen-Yu; Zen, Ke

    2014-01-01

    Evidence of a causal link between male obesity and subfertility or infertility has been demonstrated previously. However, the mechanism underlying this link is incompletely understood. Here, we report that sustained high protein-tyrosine phosphatase 1B (PTP1B) activity in sperm of obese donors plays an essential role in coupling male obesity and subfertility or infertility. First, PTP1B level and activity were significantly higher in sperm from ob/ob mice than in wild-type littermates. High PTP1B level and activity in sperm was also observed in obese patients compared with non-obese donors. The enhanced sperm PTP1B level and activity in ob/ob mice and obese patients correlated with a defect of the sperm acrosome reaction (AR). Second, treating sperm from male ob/ob mice or obese men with a specific PTP1B inhibitor largely restored the sperm AR. Finally, blockade of sperm AR by enhanced PTP1B activity in male ob/ob mice or obese men was due to prolonged dephosphorylation of N-ethylmaleimide-sensitive factor by PTP1B, leading to the inability to reassemble the trans-SNARE complexes, which is a critical step in sperm acrosomal exocytosis. In summary, our study demonstrates for the first time that a sustained high PTP1B level or activity in the sperm of obese donors causes a defect of sperm AR and that PTP1B is a novel potential therapeutic target for male infertility treatment. PMID:24519936

  3. A defined medium supports changes consistent with capacitation in stallion sperm, as evidenced by increases in protein tyrosine phosphorylation and high rates of acrosomal exocytosis.

    PubMed

    McPartlin, L A; Littell, J; Mark, E; Nelson, J L; Travis, A J; Bedford-Guaus, S J

    2008-03-15

    Efficient in vitro capacitation of stallion sperm has not yet been achieved, as suggested by low sperm penetration rates reported in in vitro fertilization (IVF) studies. Our objectives were to evaluate defined incubation conditions that would support changes consistent with capacitation in stallion sperm. Protein tyrosine phosphorylation events and the ability of sperm to undergo acrosomal exocytosis under various incubation conditions were used as end points for capacitation. Sperm incubated 4-6h in modified Whitten's (MW) with the addition of 25 mM NaHCO3 and 7 mg/mL BSA (capacitating medium) yielded high rates of protein tyrosine phosphorylation. Either HCO3(-) or BSA was required to support these changes, with the combination of both providing the most intense results. When a membrane-permeable form of cAMP and a phosphodiesterase inhibitor (IBMX) were added to MW in the absence of HCO3(-) and BSA, the tyrosine phosphorylation results obtained in our capacitating conditions could not be replicated, suggesting either effects apart from cAMP were responsible for tyrosine phosphorylation, or that stallion sperm might respond differently to these reagents as compared to sperm from other mammals. Sperm incubation in capacitating conditions was also associated with high percentages (Pacrosomal exocytosis upon exposure to progesterone (44.6%) or calcium ionophore (51.6%), as compared to sperm incubated in medium devoid of BSA and NaHCO3. Our results were novel in that we report protein tyrosine phosphorylation in stallion sperm incubated in defined conditions, coupled with significant percentages of acrosome reacted sperm. The continuation of these studies might help to elucidate the conditions and pathways supporting sperm capacitation in the horse.

  4. Changes in subcellular elemental distributions accompanying the acrosome reaction in sea urchin sperm

    SciTech Connect

    Cantino, M.E.; Schackmann, R.W.; Johnson, D.E.

    1983-05-01

    Energy-dispersive x-ray microanalysis was used to analyze changes in the subcellular distributions of Na, Mg, P, S, Cl, K, and Ca associated with the acrosome reaction of sea urchin sperm. Within 5 sec after induction of the acrosome reaction, nuclear Na and mitochondrial Ca increased and nuclear and mitochondrial K decreased. Uptake of mitochondrial P was detected after several minutes, and increases in nuclear Mg were detected only after 5-10 min of incubation following induction of the reaction. The results suggest that sudden permeability changes in the sperm plasma membrane are associated with the acrosome reaction, but that complete breakdown of membrane and cell function does not occur for several minutes.

  5. Calcium channel antagonists inhibit the acrosome reaction and bind to plasma membranes of sea urchin sperm.

    PubMed Central

    Kazazoglou, T; Schackmann, R W; Fosset, M; Shapiro, B M

    1985-01-01

    As a prerequisite to fertilization, sea urchin sperm undergo an acrosome reaction that is mediated in part by increased permeability to Ca2+, with an attendant rapid, massive intracellular Ca2+ accumulation. The acrosome reaction is inhibited by Ca2+ channel antagonists, including verapamil, D600, and dihydropyridines such as nitrendipine, nimodipine, and nisoldipine. To examine the interaction of Ca2+ antagonists with sperm, a plasma membrane preparation enriched for Na+,K+-ATPase was isolated from sea urchin sperm. These plasma membranes specifically bound [3H]nitrendipine and [3H]verapamil at concentrations similar to those that inhibit the acrosome reaction. The binding of verapamil was sigmoidal and half-maximal at 1 microM. There was a high specificity in the binding interaction, since by competition binding verapamil, (-)-D600, and (+)-D600 had different relative Kd values, 11, 2.5, and 0.5 microM, respectively. These data suggest that sperm mediate the Ca2+ influx required for induction of the acrosome reaction via Ca2+ channels with properties similar, but not identical, to those of other excitable tissues. Images PMID:3856274

  6. Lectin staining and flow cytometry reveals female-induced sperm acrosome reaction and surface carbohydrate reorganization

    PubMed Central

    Kekäläinen, Jukka; Larma, Irma; Linden, Matthew; Evans, Jonathan P.

    2015-01-01

    All cells are covered by glycans, an individually unique layer of oligo- and polysaccharides that are critical moderators of self-recognition and other cellular-level interactions (e.g. fertilization). The functional similarity between these processes suggests that gamete surface glycans may also have an important, but currently overlooked, role in sexual selection. Here we develop a user-friendly methodological approach designed to facilitate future tests of this possibility. Our proposed method is based on flow cytometric quantification of female-induced sperm acrosome reaction and sperm surface glycan modifications in the Mediterranean mussel Mytilus galloprovincialis. In this species, as with many other taxa, eggs release water-soluble factors that attract conspecific sperm (chemoattraction) and promote potentially measurable changes in sperm behavior and physiology. We demonstrate that flow cytometry is able to identify sperm from other seawater particles as well as accurately measure both acrosome reaction and structural modifications in sperm glycans. This methodological approach can increase our understanding of chemically-moderated gamete-level interactions and individual-specific gamete recognition in Mytilus sp. and other taxa with similar, easily identifiable acrosome structure. Our approach is also likely to be applicable to several other species, since carbohydrate-mediated cellular-level interactions between gametes are universal among externally and internally fertilizing species. PMID:26470849

  7. Presence and Function of Dopamine Transporter (DAT) in Stallion Sperm: Dopamine Modulates Sperm Motility and Acrosomal Integrity

    PubMed Central

    Covarrubias, Alejandra A.; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I.

    2014-01-01

    Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP+), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility. PMID:25402186

  8. Presence and function of dopamine transporter (DAT) in stallion sperm: dopamine modulates sperm motility and acrosomal integrity.

    PubMed

    Urra, Javier A; Villaroel-Espíndola, Franz; Covarrubias, Alejandra A; Rodríguez-Gil, Joan Enric; Ramírez-Reveco, Alfredo; Concha, Ilona I

    2014-01-01

    Dopamine is a catecholamine with multiple physiological functions, playing a key role in nervous system; however its participation in reproductive processes and sperm physiology is controversial. High dopamine concentrations have been reported in different portions of the feminine and masculine reproductive tract, although the role fulfilled by this catecholamine in reproductive physiology is as yet unknown. We have previously shown that dopamine type 2 receptor is functional in boar sperm, suggesting that dopamine acts as a physiological modulator of sperm viability, capacitation and motility. In the present study, using immunodetection methods, we revealed the presence of several proteins important for the dopamine uptake and signalling in mammalian sperm, specifically monoamine transporters as dopamine (DAT), serotonin (SERT) and norepinephrine (NET) transporters in equine sperm. We also demonstrated for the first time in equine sperm a functional dopamine transporter using 4-[4-(Dimethylamino)styryl]-N-methylpyridinium iodide (ASP(+)), as substrate. In addition, we also showed that dopamine (1 mM) treatment in vitro, does not affect sperm viability but decreases total and progressive sperm motility. This effect is reversed by blocking the dopamine transporter with the selective inhibitor vanoxerine (GBR12909) and non-selective inhibitors of dopamine reuptake such as nomifensine and bupropion. The effect of dopamine in sperm physiology was evaluated and we demonstrated that acrosome integrity and thyrosine phosphorylation in equine sperm is significantly reduced at high concentrations of this catecholamine. In summary, our results revealed the presence of monoamine transporter DAT, NET and SERT in equine sperm, and that the dopamine uptake by DAT can regulate sperm function, specifically acrosomal integrity and sperm motility.

  9. An egg envelope component induces the acrosome reaction in sturgeon sperm.

    PubMed

    Cherr, G N; Clark, W H

    1985-04-01

    The acrosome reaction in Acipenser transmontanus sperm can be induced by a 66,000 dalton glycoprotein that is present in Layer 3 (L3) of the egg envelope and in egg water only following exposure of the eggs to fresh water. When egg water is fractionated on Sepharose CL 6B, the 66,000 dalton glycoprotein-containing fractions possess acrosome reaction inducing activity. Egg water may be species-specific in its ability to elicit the acrosome reaction, as demonstrated by the fact that it has no effect on the sperm of Acipenser fulvescens. Egg jelly possesses no acrosome reaction, inducing activity. The major carbohydrate-containing component of the egg envelope is L3, a layer that contains galactose residues. L3 possesses a 70,000 dalton glycoprotein prior to freshwater exposure and lacks the 66,000 dalton component. If isolated from polyacrylamide gels, the 70,000 dalton glycoprotein elicits acrosome reactions in what appears to be a species specific manner. After freshwater exposure, L3 contains both the 70,000 dalton glycoprotein and the 66,000 dalton glycoprotein that is also present in egg water. The appearance of the 66,000 dalton inducer can be blocked by the incubation of eggs in fresh water containing inhibitors of trypsin activity. Thus, the soluble inducer in egg water may be proteolytically derived from a higher molecular weight complex in the egg envelope.

  10. The role and importance of cofilin in human sperm capacitation and the acrosome reaction.

    PubMed

    Megnagi, Bar; Finkelstein, Maya; Shabtay, Ortal; Breitbart, Haim

    2015-12-01

    The spermatozoon is capable of fertilizing an oocyte only after undergoing several biochemical changes in the female reproductive tract, referred to as capacitation. The capacitated spermatozoon interacts with the egg zona pellucida and undergoes the acrosome reaction, which enables its penetration into the egg and fertilization. Actin dynamics play a major role throughout all these processes. Actin polymerization occurs during capacitation, whereas prior to the acrosome reaction, F-actin must undergo depolymerization. In the present study, we describe the presence of the actin-severing protein, cofilin, in human sperm. We examined the function and regulation of cofilin during human sperm capacitation and compared it to gelsolin, an actin-severing protein that was previously investigated by our group. In contrast to gelsolin, we found that cofilin is mainly phosphorylated/inhibited at the beginning of capacitation, and dephosphorylation occurs towards the end of the process. In addition, unlike gelsolin, cofilin phosphorylation is not affected by changing the cellular levels of PIP2. Despite the different regulation of the two proteins, the role of cofilin appears similar to that of gelsolin, and its activation leads to actin depolymerization, inhibition of sperm motility and induction of the acrosome reaction. Moreover, like gelsolin, cofilin translocates from the tail to the head during capacitation. In summary, gelsolin and cofilin play a similar role in F-actin depolymerization prior to the acrosome reaction but their pattern of phosphorylation/inactivation during the capacitation process is different. Thus, for the sperm to achieve high levels of F-actin along the capacitation process, both proteins must be inactivated at different times and, in order to depolymerize F-actin, both must be activated prior to the acrosome reaction.

  11. Zn2+-stimulation of sperm capacitation and of the acrosome reaction is mediated by EGFR activation.

    PubMed

    Michailov, Yulia; Ickowicz, Debbi; Breitbart, Haim

    2014-12-15

    Extracellular zinc regulates cell proliferation via the MAP1 kinase pathway in several cell types, and has been shown to act as a signaling molecule. The testis contains a relatively high concentration of Zn(2+), required in both the early and late stages of spermatogenesis. Despite the clinical significance of this ion, its role in mature sperm cells is poorly understood. In this study, we characterized the role of Zn(2+) in sperm capacitation and in the acrosome reaction. Western blot analysis revealed the presence of ZnR of the GPR39 type in sperm cells. We previously demonstrated the presence of active epidermal growth factor receptor (EGFR) in sperm, its possible transactivation by direct activation of G-protein coupled receptor (GPCR), and its involvement in sperm capacitation and in the acrosome reaction (AR). We show here that Zn(2+) activates the EGFR during sperm capacitation, which is mediated by activation of trans-membrane adenylyl cyclase (tmAC), protein kinase A (PKA), and the tyrosine kinase, Src. Moreover, the addition of Zn(2+) to capacitated sperm caused further stimulation of EGFR and phosphatydil-inositol-3-kinase (PI3K) phosphorylation, leading to the AR. The stimulation of the AR by Zn(2+) also occurred in the absence of Ca(2+) in the incubation medium, and required the tmAC, indicating that Zn(2+) activates a GPCR. The AR stimulated by Zn(2+) is mediated by GPR39 receptor, PKA, Src and the EGFR, as well as the EGFR down-stream effectors PI3K, phospholipase C (PLC) and protein kinase C (PKC). These data support a role for extracellular zinc, acting through the ZnR, in regulating multiple signaling pathways in sperm capacitation and the acrosome reaction.

  12. Phosphoinositide-Dependent Pathways in Mouse Sperm are Regulated by Egg ZP3 and Drive the Acrosome Reaction

    PubMed Central

    Jungnickel, Melissa K.; Sutton, Keith A.; Wang, Yanli; Florman, Harvey M.

    2007-01-01

    Sperm of many animals must complete an exocytotic event, the acrosome reaction, in order to fuse with eggs. In mammals, acrosome reactions are triggered during sperm contact with the egg extracellular matrix, or zona pellucida, by the matrix glycoprotein ZP3. Here, we show that ZP3 stimulates production of phosphatidylinositol-(3,4,5)-triphosphate in sperm membranes. Phosphatidylinositol-3-kinase antagonists that prevent the production of this phosphoinositide blocked acrosome reactions and fertilization in vitro, while generation of this phosphoinositide in the absence of ZP3 triggered acrosome reactions. Downstream effectors of phosphatidylinositol-(3,4,5)-triphosphate in sperm include the protein kinases, Akt and PKCζ. These studies outline a signal transduction pathway that plays an essential role in the early events of mammalian fertilization. PMID:17258189

  13. ZP3-dependent activation of sperm cation channels regulates acrosomal secretion during mammalian fertilization

    PubMed Central

    1996-01-01

    The sperm acrosome reaction is a Ca(2+)-dependent secretory event required for fertilization. Adhesion to the egg's zona pellucida promotes Ca2+ influx through voltage-sensitive channels, thereby initiating secretion. We used potentiometric fluorescent probes to determine the role of sperm membrane potential in regulating Ca2+ entry. ZP3, the glycoprotein agonist of the zona pellucida, depolarizes sperm membranes by activating a pertussis toxin-insensitive mechanism with the characteristics of a poorly selective cation channel. ZP3 also activates a pertussis toxin-sensitive pathway that produces a transient rise in internal pH. The concerted effects of depolarization and alkalinization open voltage-sensitive Ca2+ channels. These observations suggest that mammalian sperm utilize membrane potential-dependent signal transduction mechanisms and that a depolarization pathway is an upstream transducing element coupling adhesion to secretion during fertilization. PMID:8707844

  14. Biogenesis of sperm acrosome is regulated by pre-mRNA alternative splicing of Acrbp in the mouse

    PubMed Central

    Kanemori, Yoshinori; Koga, Yoshitaka; Sudo, Mai; Kang, Woojin; Kashiwabara, Shin-ichi; Ikawa, Masahito; Hasuwa, Hidetoshi; Nagashima, Kiyoshi; Ishikawa, Yu; Ogonuki, Narumi; Ogura, Atsuo; Baba, Tadashi

    2016-01-01

    Proper biogenesis of a sperm-specific organelle, the acrosome, is essential for gamete interaction. An acrosomal matrix protein, ACRBP, is known as a proacrosin-binding protein. In mice, two forms of ACRBP, wild-type ACRBP-W and variant ACRBP-V5, are generated by pre-mRNA alternative splicing of Acrbp. Here, we demonstrate the functional roles of these two ACRBP proteins. ACRBP-null male mice lacking both proteins showed a severely reduced fertility, because of malformation of the acrosome. Notably, ACRBP-null spermatids failed to form a large acrosomal granule, leading to the fragmented structure of the acrosome. The acrosome malformation was rescued by transgenic expression of ACRBP-V5 in ACRBP-null spermatids. Moreover, exogenously expressed ACRBP-W blocked autoactivation of proacrosin in the acrosome. Thus, ACRBP-V5 functions in the formation and configuration of the acrosomal granule during early spermiogenesis. The major function of ACRBP-W is to retain the inactive status of proacrosin in the acrosome until acrosomal exocytosis. PMID:27303034

  15. CaMKII prevents spontaneous acrosomal exocytosis in sperm through induction of actin polymerization.

    PubMed

    Shabtay, Ortal; Breitbart, Haim

    2016-07-01

    In order to interact with the egg and undergo acrosomal exocytosis or the acrosome reaction (AR), mammalian spermatozoa must undergo a series of biochemical changes in the female reproductive tract, collectively called capacitation. We showed that F-actin is formed during sperm capacitation and fast depolymerization occurs prior to the AR. We hypothesized that F-actin protects the sperm from undergoing spontaneous-AR (sAR) which decreases fertilization rate. We show that activation of the actin-severing protein gelsolin induces a significant increase in sAR. Moreover, inhibition of CaMKII or PLD during sperm capacitation, caused an increase in sAR and inhibition of F-actin formation. Spermine, which leads to PLD activation, was able to reverse the effects of CaMKII inhibition on sAR-increase and F-actin-decrease. Furthermore, the increase in sAR and the decrease in F-actin caused by the inactivation of the PLD-pathway, were reversed by activation of CaMKII using H2O2 or by inhibiting protein phosphatase 1 which enhance the phosphorylation and oxidation states of CaMKII. These results indicate that two distinct pathways lead to F-actin formation in the sperm capacitation process which prevents the occurrence of sAR.

  16. Participation of the sperm proteasome during in vitro fertilisation and the acrosome reaction in cattle.

    PubMed

    Sánchez, R; Deppe, M; Schulz, M; Bravo, P; Villegas, J; Morales, P; Risopatrón, J

    2011-04-01

    In this work, we have investigated the role of the bovine sperm proteasome during in vitro fertilisation (IVF) and the acrosome reaction (AR). Motile spermatozoa, obtained by a swim-up method in Sperm-Talp medium, were capacitated for 3.5 h and incubated in the presence or absence of the specific proteasome inhibitor epoxomicin for 30 and 60 min. Then, the spermatozoa were co-incubated with mature bovine cumulus oocytes and after 48 h the cleavage rate of inseminated oocytes was evaluated. In addition, we evaluated the participation of the sperm proteasome during the progesterone-induced AR. Capacitated spermatozoa were incubated for 30 min with or without epoxomicin, then progesterone was added and the ARs were evaluated using the dual fluorescent staining technique 'Hoechst and chlortetracycline'. The results indicate that the proteasome inhibitor decreased the cleavage rate of oocytes inseminated with treated spermatozoa. In addition, acrosomal exocytosis levels were statistically significantly higher in the samples treated with the AR inducer progesterone than in control samples in the absence of the inducer. However, the progesterone-induced AR was significantly reduced by previous treatment of the spermatozoa with epoxomicin (P < 0.001). These observations indicate that the bovine sperm proteasome participates in the IVF and AR processes.

  17. The Calcium-mobilizing Messenger Nicotinic Acid Adenine Dinucleotide Phosphate Participates in Sperm Activation by Mediating the Acrosome Reaction*

    PubMed Central

    Vasudevan, Sridhar R.; Lewis, Alexander M.; Chan, Jennifer W.; Machin, Claire L.; Sinha, Debroshi; Galione, Antony; Churchill, Grant C.

    2010-01-01

    Before a sperm can fertilize an egg it must undergo a final activation step induced by the egg termed the acrosome reaction. During the acrosome reaction a lysosome-related organelle, the acrosome, fuses with the plasma membrane to release hydrolytic enzymes and expose an egg-binding protein. Because NAADP (nicotinic acid adenine dinucleotide phosphate) releases Ca2+ from acidic lysosome-related organelles in other cell types, we investigated a possible role for NAADP in mediating the acrosome reaction. We report that NAADP binds with high affinity to permeabilized sea urchin sperm. Moreover, we used Mn2+ quenching of luminal fura-2 and 45Ca2+ to directly demonstrate NAADP regulation of a cation channel on the acrosome. Additionally, we show that NAADP synthesis occurs through base exchange and is driven by an increase in Ca2+. We propose a new model for acrosome reaction signaling in which Ca2+ influx initiated by egg jelly stimulates NAADP synthesis and that this NAADP acts on its receptor/channel on the acrosome to release Ca2+ to drive acrosomal exocytosis. PMID:20400502

  18. Involvement of zinc in the regulation of pHi, motility, and acrosome reactions in sea urchin sperm

    PubMed Central

    1985-01-01

    When sperm of Strongylocentrotus purpuratus or Lytechinus pictus are diluted into seawater, motility is initiated; and when exposed to egg jelly, an acrosome reaction is induced. In the presence of a variety of structurally different metal chelators (0.1-1 mM EDTA, EGTA, phenanthroline, dipyridyl, cysteine, or dithiothreitol), motility initiation is delayed and the acrosome reaction is inhibited. Of the metals detected in the sperm of these two species, very low levels of Zn+2 (0.1 microM free Zn+2) uniquely prevent this chelator inhibition. L. pictus sperm concentrate 65Zn+2 from seawater, and EDTA removes 50% of the accumulated 65Zn+2 by 5 min. Since both sperm motility and acrosome reactions are in part regulated by intracellular pH (pHi), the effect of chelators on the sperm pHi was examined by using the fluorescent pH sensitive probe, 9-aminoacridine, EDTA depresses sperm pHi in both species, and 0.1 microM free Zn+2 reverses this pHi depression. When sperm are diluted into media that contain chelators, both NH4Cl and monensin (a Na+/H+ ionophore) increase the sperm pHi and reverse the chelator inhibition of sperm motility and acrosome reactions. The results of this study are consistent with the involvement of a trace metal (probably zinc) in the pHi regulation of sea urchin sperm and indicate a likely mechanism for the previously observed effects of chelators on sperm motility and acrosome reactions. PMID:3922992

  19. Proteomic Characterization of Pig Sperm Anterior Head Plasma Membrane Reveals Roles of Acrosomal Proteins in ZP3 Binding.

    PubMed

    Kongmanas, Kessiri; Kruevaisayawan, Hathairat; Saewu, Arpornrad; Sugeng, Clarissa; Fernandes, Jason; Souda, Puneet; Angel, Jonathan B; Faull, Kym F; Aitken, R John; Whitelegge, Julian; Hardy, Daniel; Berger, Trish; Baker, Mark A; Tanphaichitr, Nongnuj

    2015-02-01

    The sperm anterior head plasma membrane (APM) is the site where sperm first bind to the zona pellucida (ZP). This binding reaches the maximum following the sperm capacitation process. To gain a better understanding of the sperm-ZP binding mechanisms, we compared protein profiles obtained from mass spectrometry of APM vesicles isolated from non-capacitated and capacitated sperm. The results revealed that ZP-binding proteins were the most abundant group of proteins, with a number of them showing increased levels in capacitated sperm. Blue native gel electrophoresis and far-western blotting revealed presence of high molecular weight (HMW) protein complexes in APM vesicles of both non-capacitated and capacitated sperm, but the complexes (∼750-1300 kDa) from capacitated sperm possessed much higher binding capacity to pig ZP3 glycoprotein. Proteomic analyses indicated that a number of proteins known for their acrosome localization, including zonadhesin, proacrosin/acrosin and ACRBP, were components of capacitated APM HMW complexes, with zonadhesin being the most enriched protein. Our immunofluorescence results further demonstrated that a fraction of these acrosomal proteins was transported to the surface of live acrosome-intact sperm during capacitation. Co-immunoprecipitation indicated that zonadhesin, proacrosin/acrosin and ACRBP interacted with each other and they may traffic as a complex from the acrosome to the sperm surface. Finally, the significance of zonadhesin in the binding of APM HMW complexes to pig ZP3 was demonstrated; the binding ability was decreased following treatment of the complexes with anti-zonadhesin antibody. Our results suggested that acrosomal proteins, especially zonadhesin, played roles in the initial sperm-ZP binding during capacitation.

  20. Effects of atrazine and fenoxaprop-ethyl on capacitation and the acrosomal reaction in boar sperm.

    PubMed

    Maravilla-Galván, Ramiro; Fierro, Reyna; González-Márquez, Humberto; Gómez-Arroyo, Sandra; Jiménez, Irma; Betancourt, Miguel

    2009-01-01

    Atrazine is a herbicide of the chloro-s-triazine family. It inhibits photosynthesis in plants and is an endocrine disruptor, but its effects on human health are controversial. Fenoxaprop-ethyl, an aryloxy phenoxyalkanoic acid herbicide, inhibits the biosynthesis of fatty acids and provokes depolarization of membranes. The aim of this study is to evaluate the in vitro effects of both herbicides on capacitation, spontaneous acrosome reaction (SAR) and progesterone-induced acrosome reaction (PIAR) in boar sperm. Sperm capacitation is done in TALP-HEPES media for 4 hours. Capacitation and SAR are evaluated immediately; PIAR, 30 minutes later. LC50 for fenoxaprop-ethyl is 60 micromolar [corrected] and 40 micromolar [corrected] for atrazine. Fenoxaprop-ethyl induces capacitation at 60 micromolar [corrected] and SAR at all concentrations, also increases significantly PIAR. Atrazine decreased capacitation whereas increase significantly SAR and PIAR at all concentrations. It seems that fenoxaprop-ethyl and atrazine accelerate the capacitation and the acrosomal reaction, possibly via plasma membrane destabilization.

  1. Epac, Rap and Rab3 act in concert to mobilize calcium from sperm's acrosome during exocytosis.

    PubMed

    Ruete, María C; Lucchesi, Ornella; Bustos, Matías A; Tomes, Claudia N

    2014-08-27

    Exocytosis of sperm's single secretory granule or acrosome (acrosome reaction, AR) is a highly regulated event essential for fertilization. The AR begins with an influx of calcium from the extracellular milieu and continues with the synthesis of cAMP and the activation of its target Epac. The cascade bifurcates into a Rab3-GTP-driven limb that assembles the fusion machinery and a Rap-GTP-driven limb that mobilizes internal calcium. To understand the crosstalk between the two signaling cascades, we applied known AR inhibitors in three experimental approaches: reversible, stage-specific blockers in a functional assay, a far-immunofluorescence protocol to detect active Rab3 and Rap, and single cell-confocal microscopy to visualize fluctuations in internal calcium stores. Our model system was human sperm with their plasma membrane permeabilized with streptolysin O and stimulated with external calcium. The inhibition caused by reagents that prevented the activation of Rap was reversed by mobilizing intracellular calcium pharmacologically, whereas that caused by AR inhibitors that impeded Rab3's binding to GTP was not. Both limbs of the exocytotic cascade joined at or near the stage catalyzed by Rab3 in a unidirectional, hierarchical connection in which the intra-acrosomal calcium mobilization arm was subordinated to the fusion protein arm; somewhere after Rab3, the pathways became independent. We delineated the sequence of events that connect an external calcium signal to internal calcium mobilization during exocytosis. We have taken advantage of the versatility of the sperm model to investigate how cAMP, calcium, and the proteinaceous fusion machinery coordinate to accomplish secretion. Because the requirement of calcium from two different sources is not unique to sperm and fusion proteins are highly conserved, our findings might contribute to elucidate mechanisms that operate in regulated exocytosis in other secretory cell types.

  2. p-Aminobenzamidine, an acrosin inhibitor, inhibits mouse sperm penetration of the zona pellucida but not the acrosome reaction.

    PubMed

    Fraser, L R

    1982-05-01

    The effect of p-aminobenzamidine (pAB), an inhibitor of mouse sperm acrosin, on mouse sperm capacitation, motility, acrosome loss and fertility in vitro was examined using zona-intact and zona-free eggs. With intact eggs, concentrations of pAB ranging from 0.1 to 1.0 mM in the sperm preincubation medium effectively inhibited fertilization (13-0%, respectively), but these same suspensions (10(6) cells/ml) showed high rates of fertilization with zona-free eggs (100-95.3%); with the lower concentration of 10(5) cells/ml, fertilization rates of zona-free eggs decreased with increasing concentrations of pAB (100-55%). Washing of treated samples gave fertilization rates similar to control samples (87.1 and 84.6%, respectively), indicating that inhibition was reversible and that there had been no interference with the capacitation process. Whiplash motility was also observed in all samples, suggesting that the apparent inability to penetrate the zona might be due to an acrosomal defect. This was confirmed by electron microscopic examination of treated sperm samples. In high concentrations of pAB, many cells had undergone the acrosome reaction, i.e. membrane vesiculation, but acrosomal matrix dispersal was inhibited. These results are consistent, therefore, with a role for the acrosomal enzyme acrosin in matrix dispersal, but not the acrosome reaction itself, and in penetration of the zona pellucida.

  3. AM67, a secretory component of the guinea pig sperm acrosomal matrix, is related to mouse sperm protein sp56 and the complement component 4-binding proteins.

    PubMed

    Foster, J A; Friday, B B; Maulit, M T; Blobel, C; Winfrey, V P; Olson, G E; Kim, K S; Gerton, G L

    1997-05-09

    The guinea pig sperm acrosomal matrix is the dense core of the acrosome and is likely to be important in acrosome biogenesis and fertilization. Isolated acrosomal matrices are composed of a limited number of major bands when analyzed by SDS-polyacrylamide gel electrophoresis, among which is a Mr 67,000 protein that we have termed AM67. Indirect immunofluorescence demonstrated that AM67 is localized to the apical segment of the cauda epididymal sperm acrosome. Immunoelectron microscopy further refined the localization of AM67 to the M1 (dorsal bulge) domain within the acrosome. Using a polymerase chain reaction product based upon tryptic peptide sequences from AM67, a lambdagt11 guinea pig testis cDNA library was screened to yield two cDNA clones that encode the AM67 peptides. Northern analysis revealed that AM67 is transcribed as a 1. 9-kilobase testis-specific mRNA. The complete AM67 sequence encodes a prepropolypeptide of 533 amino acids with a calculated Mr of 59, 768. Following cleavage of a probable signal sequence, the polypeptide was predicted to have a Mr of 56,851 and seven consensus sites for asparagine-linked glycosylation. The deduced amino acid sequence of AM67 is most similar to those of the mouse sperm protein sp56 and the alpha-subunits of complement component 4-binding proteins from various mammalian species. Although mouse sp56 has been reported to be a cell-surface receptor for the murine zona pellucida glycoprotein ZP3, standard immunoelectron microscopy using the anti-sp56 monoclonal antibody 7C5 detected sp56 within the mouse sperm acrosome, but failed to detect sp56 on the surface of acrosome-intact mouse sperm. Furthermore, acrosomal labeling was detected in mouse sperm prepared for immunofluorescence using paraformaldehyde fixation, but was not observed with live unfixed sperm. Thus, the finding that sp56 is present within the acrosome provides further support that sp56 and AM67 are orthologues and suggests that sp56 may function in

  4. Evidence that aggregation of mouse sperm receptors by ZP3 triggers the acrosome reaction

    PubMed Central

    1989-01-01

    In the mouse, considerable evidence indicates that initial sperm binding to the zona pellucida (ZP) is mediated by ZP3. In addition, this same glycoprotein is also responsible for inducing the acrosome reaction (AR). Whereas the O-linked oligosaccharides of ZP3 appear to mediate sperm-ZP binding, the portion of ZP3 bearing AR activity has not been defined. To try to understand the bifunctional role of ZP3 (binding and AR inducing activities), we have examined the hypothesis that ZP3 aggregates sperm receptor molecules. By analogy with findings in a variety of other extracellular signal transducing systems, including receptors for growth factors and insulin, this aggregation event could initiate the cascade resulting in the AR. To test this hypothesis, we have generated monospecific polyclonal antibodies against ZP2 and against ZP3, and examined the effects of these probes on capacitated sperm incubated in the absence or presence of various ZP protein preparations. For some experiments, we have used proteolytic fragments of ZP3, a preparation known to retain specific binding, but not AR-inducing, activity. We show here that capacitated mouse sperm, incubated with ZP glycopeptides, displayed ARs when incubated subsequently with anti-ZP3 IgG; ARs did not occur when parallel sperm samples were incubated with anti-ZP2 IgG or with anti-ZP3 Fab fragments. When capacitated sperm were treated successively, with (a) ZP3 glycopeptides, (b) anti-ZP3 Fab fragments, and (c) goat anti-rabbit IgG, ARs occurred in the majority of sperm. An alternative approach to examine this hypothesis used ZP proteins obtained from tubal eggs treated previously with bioactive phorbol diester (12-O-tetradecanoyl phorbol-13-acetate [TPA]). This preparation arrests capacitated sperm in an intermediate state of the AR. We demonstrate here that these sperm can be induced to undergo a complete AR by subsequent treatment with anti-ZP3 IgG. Together, these findings are consistent with the hypothesis

  5. Ubiquitin-activating enzyme (UBA1) is required for sperm capacitation, acrosomal exocytosis and sperm-egg coat penetration during porcine fertilization.

    PubMed

    Yi, Y-J; Zimmerman, S W; Manandhar, G; Odhiambo, J F; Kennedy, C; Jonáková, V; Maňásková-Postlerová, P; Sutovsky, M; Park, C-S; Sutovsky, P

    2012-04-01

    Protein ubiquitination is a stable, covalent post-translational modification that alters protein activity and/or targets proteins for proteolysis by the 26S proteasome. The E1-type ubiquitin-activating enzyme (UBA1) is responsible for ubiquitin activation, the initial step of ubiquitin-protein ligation. Proteasomal proteolysis of ubiquitinated spermatozoa and oocyte proteins occurs during mammalian fertilization, particularly at the site of sperm acrosome contact with oocyte zona pellucida. However, it is not clear whether the substrates are solely proteins ubiquitinated during gametogenesis or if de novo ubiquitination also occurs during fertilization supported by ubiquitin-activating and -conjugating enzymes present in the sperm acrosome. Along this line of inquiry, UBA1 was detected in boar sperm-acrosomal extracts by Western blotting (WB). Immunofluorescence revealed accumulation of UBA1 in the nuclei of spermatogonia, spermatocytes and spermatids, and in the acrosomal caps of round and elongating spermatids. Thiol ester assays utilizing biotinylated ubiquitin and isolated sperm acrosomes confirmed the enzymatic activity of the resident UBA1. A specific UBA1 inhibitor, PYR-41, altered the remodelling of the outer acrosomal membrane (OAM) during sperm capacitation, monitored using flow cytometry of fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA). Although viable and motile, the spermatozoa capacitated in the presence of PYR-41, showed significantly reduced fertilization rates during in vitro fertilization (IVF; p < 0.05). Similarly, the fertilization rate was lowered by the addition of PYR-41 directly into fertilization medium during IVF. In WB, high Mr bands, suggestive of protein ubiquitination, were detected in non-capacitated spermatozoa by antibodies against ubiquitin; WB with anti-phosphotyrosine antibodies and antibodies against acrosomal proteins SPINK2 (acrosin inhibitor) and AQN1 (spermadhesin) revealed that the capacitation

  6. Osmotic tolerance limits and effects of cryoprotectants on the motility, plasma membrane integrity and acrosomal integrity of rat sperm.

    PubMed

    Si, Wei; Benson, James D; Men, Hongsheng; Critser, John K

    2006-12-01

    Osmotic stress is an important factor that can result in cell damage during cryopreservation. The objectives of this study were to determine: (1) isosmotic sperm cell volume; (2) osmotically inactive volume; (3) osmotic tolerance limits of rat sperm; and (4) the effects of addition and removal of glycerol (Gly), ethylene glycol (EG), propylene glycol (PG) or dimethyl sulfoxide (Me(2)SO) on rat sperm function. Sperm from Fischer 344 and Sprague-Dawley rats were used in this study. An electronic particle counter was used to measure the cell volume of rat sperm. Computer-assisted sperm motility analysis and flow-cytometric analysis were used to assess sperm motility, plasma membrane and acrosomal integrity. The isosmotic sperm cell volumes of the two strains were 37.0+/-0.1 and 36.2+/-0.2 microm(3), respectively. Rat sperm behaved as linear osmometers from 260 to 450 mOsm, and the osmotically inactive sperm volumes of the two strains were 79.8+/-1.5% and 81.4+/-2.2%, respectively. Rat sperm have very limited osmotic tolerances. The sperm motility and the sperm plasma membranes of both strains were sensitive to anisosmotic treatments, but the acrosomes of both strains were more sensitive to hyposmotic than hyperosmotic conditions. The one-step addition and removal of Me(2)SO showed the most deleterious effect on rat sperm motility, plasma membrane integrity, and acrosomal integrity among the four cryoprotectants. These data characterizing rat sperm osmotic behavior, osmotic and cryoprotectant tolerance will be used to design cryopreservation protocols for rat sperm.

  7. Fluorometric study of rabbit sperm head membrane phospholipid asymmetry during capacitation and acrosome reaction using Annexin-V FITC.

    PubMed

    Avalos-Rodríguez, A; Ortíz-Muñíz, A R; Ortega-Camarillo, C; Vergara-Onofre, M; Rosado-García, A; Rosales-Torres, A M

    2004-01-01

    This study was conducted to evaluate phosphatidylserine translocation in head plasma membrane of Percoll-gradient purified of rabbit cauda epididymal sperm during capacitation and acrosome reaction (AR) using Annexin-V. Propidium iodide was used as control to reject dead or dying cells. The presence and distribution of Annexin-V binding sites were analyzed using flow fluorocytometry and confocal microscopy. After 6 h of incubation of sperm in capacitation medium, the number of cells positively stained with Annexin-V showed a small but significant increment. The Annexin-V binding sites produced during capacitation were found mainly in the post-acrosomal region of the sperm head plasma membrane. After AR induction with progesterone, the localization of phosphatidylserine was changed and the Annexin-V binding sites were found almost only in the acrosomal region, but with higher number of binding sites in the equatorial area. On the contrary, after AR induction with A23187, phosphatidylserine translocation, although predominant over the acrosomal region, was also observed in the post-acrosomal region. Plasma membrane destabilization during capacitation and AR may be important for sperm-oocyte fusion.

  8. Expression of a P-selectin ligand in zona pellucida of porcine oocytes and P-selectin on acrosomal membrane of porcine sperm cells. Potential implications for their involvement in sperm-egg interactions.

    PubMed

    Geng, J G; Raub, T J; Baker, C A; Sawada, G A; Ma, L; Elhammer, A P

    1997-05-05

    The selectin family of cell adhesion molecules mediates initial leukocyte adhesion to vascular endothelial cells at sites of inflammation. O-glycan structural similarities between oligosaccharides from human leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) and from zona pellucida glycoproteins of porcine oocytes indicate the possible existence of a P-selectin ligand in the zona pellucida. Here, using biochemical as well as morphological approaches, we demonstrate that a P-selectin ligand is expressed in the porcine zona pellucida. In addition, a search for a specific receptor for this ligand leads to the identification of P-selectin on the acrosomal membrane of porcine sperm cells. In vitro binding of porcine acrosome-reacted sperm cells to oocytes was found to be Ca2+ dependent and inhibitable with either P-selectin, P-selectin receptor-globulin, or leukocyte adhesion blocking antibodies against P-selectin and PSGL-1. Moreover, porcine sperm cells were found to be capable of binding to human promyeloid cell line HL-60. Taken together, our findings implicate a potential role for the oocyte P-selectin ligand and the sperm P-selectin in porcine sperm-egg interactions.

  9. Validation of non-fluorescent methods to reliably detect acrosomal and plasma membrane integrity of common marmoset (Callithrix jacchus) sperm.

    PubMed

    Valle, R R; Valle, C M R; Nichi, M; Muniz, J A P C; Nayudu, P L; Guimarães, M A B V

    2008-07-01

    Simple, rapid and stable sperm evaluation methods which have been optimized for common marmoset (Callithrix jacchus) are critical for studies involving collection and evaluation of sperm in the field. This is particularly important for new species groups such as Callitrichidae where the sperm have been little studied. Of this family, C. jacchus is the best known, and has been chosen as a model species for other members of the genus Callithrix. The fundamental evaluation parameters for sperm of any species are viability and acrosomal status. Semen samples were collected by penile vibratory stimulation. To evaluate sperm plasma membrane integrity, Eosin-Nigrosin was tested here for the common marmoset sperm to be used under field conditions. Further, a non-fluorescent stain for acrosome, the "Simple" stain, developed for domestic and wild cats, was tested on common marmoset sperm. This was compared with a fluorescent staining, Fluorescein isothiocyanate-Pisum sativum agglutinin (FITC-PSA), routinely used and validated for common marmoset at the German Primate Centre to evaluate acrosomal integrity. Results obtained with the "Simple" stain showed a marked differentiation between sperm with intact and non-intact acrosome both with and without ionophore treatment and closely correlated with results obtained with FITC-PSA. Temperature had no effect on the results with the "Simple" stain and the complete processing is simple enough to be carried out under field conditions. These findings indicated that the "Simple" stain and Eosin-Nigrosin provide rapid and accurate results for C. jacchus sperm and that those methods can be reliably used as field tools for sperm evaluation for this species.

  10. A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm.

    PubMed

    Romarowski, Ana; Sánchez-Cárdenas, Claudia; Ramírez-Gómez, Héctor V; Puga Molina, Lis del C; Treviño, Claudia L; Hernández-Cruz, Arturo; Darszon, Alberto; Buffone, Mariano G

    2016-03-01

    During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca(2+) ([Ca(2+)]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca(2+)]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca(2+)]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca(2+)]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca(2+)]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca(2+)]i originating in the sperm head promotes the initiation of AR. © 2016 by the Society for the Study of Reproduction, Inc.

  11. Effect of washing and capacitating media pH on bull sperm motility, acrosome integrity, and ability to penetrate zona-free hamster oocytes.

    PubMed

    Ijaz, A; Hunter, A G

    1989-10-01

    Bovine-ejaculated sperm were washed thrice in bovine serum albumin-saline media, pH 7.2 to 8.4, and incubated at 37 degrees C in Ca++-free Tyrode's media. pH 7.2 to 8.4, for 0, 2, 4, 6, and 8 h. Motility was highest when sperm were washed in pH 7.2 medium and incubated in pH 8.0 or 8.4 media. Motility remained above 50% until 8 h. Washing in pH 7.6, 8.0, or 8.4 media induced more acrosome reactions after incubation than washing at pH 7.2. Percentage of acrosome-reacted sperm increased at each successive time period. Sperm penetrated more oocytes at 4, 6, and 8 h when wash medium pH was fixed at 7.2 and capacitating media pH was raised at .4 unit increments from 7.2 to 8.4. When sperm were washed in pH 7.2 medium, the postincubation penetration rates peaked at 8 h. With wash media of pH 7.6, 8.0, or 8.4, the postincubation penetration rates peaked at 4 h and then gradually declined. In conclusion, the most effective system for capacitating bull sperm was a pH 7.6 wash followed by capacitation in pH 7.6 medium for 4 to 8 h and this system resulted in the highest penetration rates. Wash media pH hastened capacitation but was not a capacitating agent.

  12. Regulation of Sperm Capacitation and the Acrosome Reaction by PIP 2 and Actin Modulation.

    PubMed

    Breitbart, Haim; Finkelstein, Maya

    2015-01-01

    Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. Actin polymerization occurs during capacitation and prior to the acrosome reaction, fast F-actin breakdown takes place. The increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP 2 ) and its phosphorylation on tyrosine-438 by Src. Activation of gelsolin following its release from PIP 2 is known to cause F-actin breakdown and inhibition of sperm motility, which can be restored by adding PIP 2 to the cells. Reduction of PIP 2 synthesis inhibits actin polymerization and motility, while increasing PIP 2 synthesis enhances these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP 2 and F-actin. During capacitation there was an increase in PIP 2 and F-actin levels in the sperm head and a decrease in the tail. In spermatozoa with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends upon its binding to PIP 2 . Stimulation of phospholipase C, by Ca 2 + -ionophore or by activating the epidermal-growth-factor-receptor, inhibits tyrosine phosphorylation of gelsolin and enhances enzyme activity. In conclusion, these data indicate that the increase of PIP 2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result, the decrease of gelsolin in the tail allows the maintenance of high levels of F-actin in this structure, which is essential for the development of HA motility.

  13. Regulation of sperm capacitation and the acrosome reaction by PIP2 and actin modulation

    PubMed Central

    Breitbart, Haim; Finkelstein, Maya

    2015-01-01

    Actin polymerization and development of hyperactivated (HA) motility are two processes that take place during sperm capacitation. Actin polymerization occurs during capacitation and prior to the acrosome reaction, fast F-actin breakdown takes place. The increase in F-actin during capacitation depends upon inactivation of the actin severing protein, gelsolin, by its binding to phosphatydilinositol-4, 5-bisphosphate (PIP2) and its phosphorylation on tyrosine-438 by Src. Activation of gelsolin following its release from PIP2 is known to cause F-actin breakdown and inhibition of sperm motility, which can be restored by adding PIP2 to the cells. Reduction of PIP2 synthesis inhibits actin polymerization and motility, while increasing PIP2 synthesis enhances these activities. Furthermore, sperm demonstrating low motility contained low levels of PIP2 and F-actin. During capacitation there was an increase in PIP2 and F-actin levels in the sperm head and a decrease in the tail. In spermatozoa with high motility, gelsolin was mainly localized to the sperm head before capacitation, whereas in low motility sperm, most of the gelsolin was localized to the tail before capacitation and translocated to the head during capacitation. We also showed that phosphorylation of gelsolin on tyrosine-438 depends upon its binding to PIP2. Stimulation of phospholipase C, by Ca2+-ionophore or by activating the epidermal-growth-factor-receptor, inhibits tyrosine phosphorylation of gelsolin and enhances enzyme activity. In conclusion, these data indicate that the increase of PIP2 and/or F-actin in the head during capacitation enhances gelsolin translocation to the head. As a result, the decrease of gelsolin in the tail allows the maintenance of high levels of F-actin in this structure, which is essential for the development of HA motility. PMID:25966627

  14. Intracellular sodium changes during the speract response and the acrosome reaction in sea urchin sperm

    PubMed Central

    Rodríguez, Esmeralda; Darszon, Alberto

    2003-01-01

    The sperm-activating peptide speract and fucose-sulphate glycoconjugate (FSG) are sea urchin egg-envelope components that modulate sperm ion permeability. They influence motility and induce acrosomal reaction (AR), respectively. A fluorescent Na+-sensitive dye (Na+-binding benzofuran isophthalate, SBFI) was used to determine how these egg envelope components influence sperm Na+ permeability. [Ca2+]i and pHi were also measured to correlate their changes in response to speract and FSG with those observed in [Na+]i. SBFI determinations indicate that the resting [Na+]i is 20 ± 8 mm in sea urchin sperm. Saturating levels of speract increased [Na+]i by ≈15 mm, while similar levels of FSG caused a further elevation of ≈30 mm. The kinetics of the [Na+]i, [Ca2+]i and pHi changes induced by saturating levels of speract were faster than those induced by FSG. Both egg ligands appeared to activate more than one Na+ transport system. Nifedipine, Ni2+ and TEA+ inhibited the ionic changes and the AR induced by FSG but, importantly, did not alter those caused by speract. Thus, there are differences in some of the ionic transport mechanisms that operate in the speract and FSG responses. ZD2788, a blocker of hyperpolarization and cyclic-nucleotide-gated (HCN) channels such as SpHCN present in sea urchin sperm, did not decrease the speract-induced [Na+]i increase, but slowed its kinetics. Therefore, SpHCN does not play a major role in the uptake of Na+ triggered by this decapeptide. KB-R7943, an inhibitor of Na+/Ca2+ exchangers, decreased the resting [Na+]i and did not change significantly the speract-induced [Ca2+]i increase, but slowed its recovery. PMID:12509481

  15. Mouse sperm patch-clamp recordings reveal single Cl- channels sensitive to niflumic acid, a blocker of the sperm acrosome reaction.

    PubMed

    Espinosa, F; de la Vega-Beltrán, J L; López-González, I; Delgado, R; Labarca, P; Darszon, A

    1998-04-10

    Ion channels lie at the heart of gamete signaling. Understanding their regulation will improve our knowledge of sperm physiology, and may lead to novel contraceptive strategies. Sperm are tiny (approximately 3 microm diameter) and, until now, direct evidence of ion channel activity in these cells was lacking. Using patch-clamp recording we document here, for the first time, the presence of cationic and anionic channels in mouse sperm. Anion selective channels were blocked by niflumic acid (NA) (IC50 = 11 microM). The blocker was effective also in inhibiting the acrosome reaction induced by the zona pellucida, GABA or progesterone. These observations suggest that Cl- channels participate in the sperm acrosome reaction in mammals.

  16. Role and regulation of EGFR in actin remodeling in sperm capacitation and the acrosome reaction

    PubMed Central

    Breitbart, Haim; Etkovitz, Nir

    2011-01-01

    To bind and fertilize the egg, the spermatozoon should undergo few biochemical and motility changes in the female reproductive tract collectively called capacitation. The capacitated spermatozoon binds to the egg zona pellucida, and then undergoes the acrosome reaction (AR), which allows its penetration into the egg. The mechanisms regulating sperm capacitation and the AR are not completely understood. In the present review, we summarize some data regarding the role and regulation of the epidermal growth factor receptor (EGFR) in these processes. In the capacitation process, the EGFR is partially activated by protein kinase A (PKA), resulting in phospholipase D (PLD) activation and actin polymerization. Protein kinase C alpha (PKCα), which is already activated at the beginning of the capacitation, also participates in PLD activation. Further activation of the EGFR at the end of the capacitation enhances intracellular Ca2+ concentration leading to F-actin breakdown and allows the AR to take place. Under in vivo conditions, the EGFR can be directly activated by its known ligand epidermal growth factor (EGF), and indirectly by activating PKA or by transactivation mediated by G protein-coupled receptors (GPCRs) activation or by ouabain. Under physiological conditions, sperm PKA is activated mainly by bicarbonate, which activates the soluble adenylyl cyclase to produce cyclic adenosine monophosphate (cAMP), the activator of PKA. The GPCR activators angiotensin II or lysophosphatidic acid, as well as ouabain and EGF are physiological components present in the female reproductive tract. PMID:21200378

  17. Fine structure of acrosome biogenesis and of mature sperm in the bivalve molluscs Glycymeris sp. (Pteriomorphia) and Eurhomalea rufa (Heterodonta)

    NASA Astrophysics Data System (ADS)

    Guerra, Rosa; Sousa, Mário; Torres, Artur; Oliveira, Elsa; Baldaia, Luis

    2003-03-01

    Proacrosomal vesicles form during the pachytene stage, being synthetized by the Golgi complex in Glycymeris sp., and by both the Golgi and the rough endoplasmic reticulum in Eurhomalea rufa. During early spermiogenesis, a single acrosomal vesicle forms and its apex becomes linked to the plasma membrane while it migrates. In Glycymeris sp., the acrosomal vesicle then turns cap-shaped (1.8 μm) and acquires a complex substructure. In E. rufa, proacrosomal vesicles differentiate their contents while still at the premeiotic stage; as the acrosomal vesicle matures and its contents further differentiate, it elongates and becomes longer than the nucleus (3.2 μm), while the subacrosomal space develops a perforatorium. Before condensation, chromatin turns fibrillar in Glycymeris sp., whereas it acquires a cordonal pattern in E. rufa. Accordingly, the sperm nucleus of Glycymeris sp. is conical and elongated (8.3 μm), and that of E. rufa is short and ovoid (1.1 μm). In the midpiece (Glycymeris sp.: 1.1 μm; E. rufa: 0.8 μm), both species have four mitochondria encircling two linked orthogonal (Glycymeris sp.) or orthogonal and tilted (30-40°; E. rufa) centrioles. In comparison with other Arcoida species, sperm of Glycymeris sp. appear distinct due to the presence of an elongated nucleus, a highly differentiated acrosome, and four instead of five mitochondria. The same occurs with E. rufa regarding other Veneracea species, with the acrosome of the mature sperm strongly resembling that of the recent Mytilinae.

  18. Effects of ascorbic acid on sperm motility, viability, acrosome reaction and DNA integrity in teratozoospermic samples

    PubMed Central

    Fanaei, Hamed; Khayat, Samira; Halvaei, Iman; Ramezani, Vahid; Azizi, Yaser; Kasaeian, Amir; Mardaneh, Jalal; Parvizi, Mohammad Reza; Akrami, Maryam

    2014-01-01

    Background: Oxidative stress in teratozoospermic semen samples caused poor assisted reproductive techniques (ART) outcomes. Among antioxidants, ascorbic acid is a naturally occurring free radical scavenger and as such its presence assists various other mechanisms in decreasing numerous disruptive free radical processes. Objective: The main goal of this study was to evaluate potential protective effects of ascorbic acid supplementation during in vitro culture of teratozoospermic specimens. Materials and Methods: Teratozoospermic semen samples that collected from 15 volunteers were processed, centrifuged and incubated at 37oC until sperm swimmed-up. Supernatant was divided into four groups and incubated at 37oC for one hour under different experimental conditions: Control, 10 µm A23187, 600µm ascorbic acid and 10 µm A23187+600 µm ascorbic acid. After incubation sperm motility, viability, acrosome reaction, DNA damage and malondialdehyde levels were evaluated. Results: Our results indicated that after one hour incubation, ascorbic acid significantly reduced malondialdehyde level in ascorbic acid group (1.4±0.11 nmol/ml) compared to control group (1.58±0.13 nmol/ml) (p<0.001). At the end of incubation, progressive motility and viability in ascorbic acid group (64.5±8.8% and 80.3±6.4%, respectively) were significantly (p<0.05 and p<0.001, respectively) higher than the control group (54.5±6.8% and 70.9±7.3%, respectively). A23187 significantly (p<0.0001) increased acrosome reaction in A23187 group (37.3±5.6%) compared to control group (8.5±3.2%) and this effect of A23187 attenuated by ascorbic acid in ascorbic acid+A23187 group (17.2±4.4%). DNA fragmentation in ascorbic acid group (20±4.1%) was significantly (p<0.001) lower than controls (28.9±4.6%). Conclusion: In vitro ascorbic acid supplementation during teratozoospermic semen processing for ART could protect teratozoospermic specimens against oxidative stress, and it could improve ART outcome. PMID

  19. Actin filaments in the acrosomal reaction of Limulus sperm. Motion generated by alterations in the packing of the filaments

    PubMed Central

    1975-01-01

    When Limulus sperm are induced to undergo the acrosomal reaction, a process, 50 mum in length, is generated in a few seconds. This process rotates as it elongates; thus the acrosomal process literally screws through the jelly of the egg. Within the process is a bundle of filaments which before induction are coiled up inside the sperm. The filament bundle exists in three stable states in the sperm. One of the states can be isolated in pure form. It is composed of only three proteins whose molecular weights (mol wt) are 43,000, 55,000, and 95,000. The 43,000 mol wt protein is actin, based on its molecular weight, net charge, morphology, G-F transformation, and heavy meromyosin (HMM) binding. The 55,000 mol wt protein is in equimolar ratio to actin and is not tubulin, binds tenaciously to actin, and inhibits HMM binding. Evidence is presented that both the 55,000 mol wt protein and the 95,000 mol wt protein (possibly alpha-actinin) are also present in Limulus muscle. Presumably these proteins function in the sperm in holding the actin filaments together. Before the acrosomal reaction, the actin filaments are twisted over one another in a supercoil; when the reaction is completed, the filaments lie parallel to each other and form an actin paracrystal. This change in their packing appears to give rise to the motion of the acrosomal process and is under the control of the 55,000 mol wt protein and the 95,000 mol wt protein. PMID:1117029

  20. Automatic detection and segmentation of sperm head, acrosome and nucleus in microscopic images of human semen smears.

    PubMed

    Shaker, Fariba; Monadjemi, S Amirhassan; Naghsh-Nilchi, Ahmad Reza

    2016-08-01

    Manual assessment of sperm morphology is subjective and error prone so developing automatic methods is vital for a more accurate assessment. The first step in automatic evaluation of sperm morphology is sperm head detection and segmentation. In this paper a complete framework for automatic sperm head detection and segmentation is presented. After an initial thresholding step, the histogram of the Hue channel of HSV color space is used, in addition to size criterion, to discriminate sperm heads in microscopic images. To achieve an improved segmentation of sperm heads, an edge-based active contour method is used. Also a novel tail point detection method is proposed to refine the segmentation by locating and removing the midpiece from the segmented head. An algorithm is also proposed to separate the acrosome and nucleus using morphological operations. Dice coefficient is used to evaluate the segmentation performance. The proposed methods are evaluated using a publicly available dataset. The proposed method has achieved segmentation accuracy of 0.92 for sperm heads, 0.84 for acrosomes and 0.87 for nuclei, with the standard deviation of 0.05, which significantly outperforms the current state-of-the-art. Also our tail detection method achieved true detection rate of 96%. In this paper we presented a complete framework for sperm detection and segmentation which is totally automatic. It is shown that using active contours can improve the segmentation results of sperm heads. Our proposed algorithms for tail detection and midpiece removal further improved the segmentation results. The results indicate that our method achieved higher Dice coefficients with less dispersion compared to the existing solutions. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. α-SNAP Prevents Docking of the Acrosome during Sperm Exocytosis because It Sequesters Monomeric Syntaxin

    PubMed Central

    Rodríguez, Facundo; Bustos, Matías A.; Zanetti, María N.; Ruete, María C.; Mayorga, Luis S.; Tomes, Claudia N.

    2011-01-01

    α-SNAP has an essential role in membrane fusion that consists of bridging cis SNARE complexes to NSF. α-SNAP stimulates NSF, which releases itself, α-SNAP, and individual SNAREs that subsequently re-engage in the trans arrays indispensable for fusion. α-SNAP also binds monomeric syntaxin and NSF disengages the α-SNAP/syntaxin dimer. Here, we examine why recombinant α-SNAP blocks secretion in permeabilized human sperm despite the fact that the endogenous protein is essential for membrane fusion. The only mammalian organism with a genetically modified α-SNAP is the hyh mouse strain, which bears a M105I point mutation; males are subfertile due to defective sperm exocytosis. We report here that recombinant α-SNAP-M105I has greater affinity for the cytosolic portion of immunoprecipitated syntaxin than the wild type protein and in consequence NSF is less efficient in releasing the mutant. α-SNAP-M105I is a more potent sperm exocytosis blocker than the wild type and requires higher concentrations of NSF to rescue its effect. Unlike other fusion scenarios where SNAREs are subjected to an assembly/disassembly cycle, the fusion machinery in sperm is tuned so that SNAREs progress uni-directionally from a cis configuration in resting cells to monomeric and subsequently trans arrays in cells challenged with exocytosis inducers. By means of functional and indirect immunofluorescense assays, we show that recombinant α-SNAPs — wild type and M105I — inhibit exocytosis because they bind monomeric syntaxin and prevent this SNARE from assembling with its cognates in trans. Sequestration of free syntaxin impedes docking of the acrosome to the plasma membrane assessed by transmission electron microscopy. The N-terminal deletion mutant α-SNAP-(160–295), unable to bind syntaxin, affects neither docking nor secretion. The implications of this study are twofold: our findings explain the fertility defect of hyh mice and indicate that assembly of SNAREs in trans complexes is

  2. Quantification of bovine sperm separation by a swim-up method. Relationship to sperm motility, integrity of acrosomes, sperm migration in polyacrylamide gel and fertility.

    PubMed

    Parrish, J J; Foote, R H

    1987-01-01

    The number of bovine spermatozoa separated in a swim-up procedure was quantified using an electronic cell counter. In an initial test of the swim-up procedure, non-frozen sperm samples with different ratios of live to dead cells were prepared and tested for the number of spermatozoa counted by the swim-up procedure. In ejaculates from six bulls, the number of spermatozoa swimming up was related to the number of live cells present (R2 = 0.97). Next, sperm quality of frozen-thawed semen immediately after thawing was measured at 37 C by swim-up sperm count, sperm motility, spermatozoa with an intact acrosome and migration in polyacrylamide gel and then compared with the fertility of the semen used for artificial insemination. Twenty-nine ejaculates of frozen-thawed semen from 11 bulls were evaluated. Correlations with fertility were highest on an ejaculate basis for motility (r = 0.41, P = 0.05) and for swim-up sperm count (r = 0.35, P = 0.06). On a bull basis, swim-up sperm count had the highest correlation with fertility (r = 0.59, P = 0.06). In a multiple regression model to predict male fertility that included all described measures of semen quality, a R2 value of 0.69 was obtained. This is the first report showing that the ability of spermatozoa to swim out of a more dense medium (whole milk-glycerol extender) into culture media is quantitatively related to in vivo fertility.

  3. Motility and acrosomal integrity comparisons between electro-ejaculated and epididymal ram sperm after exposure to a range of anisosmotic solutions, cryoprotective agents and low temperatures.

    PubMed

    Varisli, Omer; Uguz, Cevdet; Agca, Cansu; Agca, Yuksel

    2009-02-01

    Effective ram sperm cryopreservation protocols, which would yield acceptable lambing rates following artificial insemination (AI), are currently lacking. The objectives of the current studies were to compare the effects of various anisosmotic conditions, cryoprotective agents (CPAs) and chilling on the motility and acrosomal integrity of electro-ejaculated and epididymal ram sperm. Three experiments were conducted. In experiment 1, ejaculated and epididymal ram sperm were exposed to 75, 150, 225, 600, 900 and 1200 milliosmolal (mOsm)/kg sucrose solutions, held for 5 min and then returned to isosmotic condition. Motility characteristics of sperm during exposure to each anisosmotic solutions and after returning to isosmotic conditions were determined. In experiment 2, ejaculated and epididymal ram sperm were exposed to 1M glycerol (Gly), dimethyl sulfoxide (DMSO), ethylene glycol (EG) and propylene glycol (PG) for 5 min and then returned to isosmotic conditions. Motility characteristics of sperm samples during exposure to each CPA solution and after returning to isosmotic conditions were determined. In experiment 3, effects of various temperatures on motility characteristics of ejaculated and epididymal ram sperm were determined after exposing them to three different sub-physiologic temperatures (4, 10 and 22 degrees C) for 30 min and subsequently returning them to 37 degrees C. The motility of ejaculated ram sperm was significantly more affected from anisosmotic stress than was epididymal ram sperm (P<0.05). While anisosmotic stress had no effects on acrosomal integrity of epididymal ram sperm, there was a significant reduction in acrosomal integrity for ejaculated ram sperm after the addition and removal of a 75 mOsm sucrose solution. The abrupt addition and removal of 1M Gly, DMSO, EG or PG had no effect on the motility and acrosomal integrity of epididymal ram sperm (P>0.05). However, there was a slight decrease in acrosomal integrity for ejaculated ram sperm

  4. Inhibitors of serine proteases decrease sperm penetration during porcine fertilization in vitro by inhibiting sperm binding to the zona pellucida and acrosome reaction.

    PubMed

    Beek, J; Nauwynck, H; Appeltant, R; Maes, D; Van Soom, A

    2015-11-01

    Serine proteases are involved in mammalian fertilization. Inhibitors of serine proteases can be applied to investigate at which point these enzymes exert their action. We selected two serine protease inhibitors, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF, 100 μM) and soybean trypsin inhibitor (STI, 5 μM) from Glycine max, via previous dose-response IVF experiments and sperm toxicity tests. In the present study, we evaluated how these inhibitors affect porcine fertilization in vitro as calculated on total fertilization rate, polyspermy rate, and the sperm number per fertilized oocyte of cumulus-intact, cumulus-free, and zona-free oocytes. In the control group (no inhibitor), these parameters were 86%, 49%, and 2.2 for cumulus-intact oocytes and 77%, 43%, and 2.2 for cumulus-free oocytes (6-hour gamete incubation period, 1.25 × 10(5) spermatozoa/mL). 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride and STI significantly reduced total fertilization and polyspermy rate in cumulus-intact and cumulus-free oocytes (P < 0.05). Total fertilization rates were respectively 65% and 53% (AEBSF) and 36% and 17% (STI). Inhibition rates were higher in cumulus-free oocytes than in cumulus-intact oocytes, indicating that inhibitors exerted their action after sperm passage through the cumulus. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride but not STI reduced sperm binding to the ZP. The acrosome reaction was significantly inhibited by both inhibitors. Only 40.4% (AEBSF) and 11.4% (STI) of spermatozoa completed a calcium-induced acrosome reaction compared to 86.7% of spermatozoa in the control group. There was no effect on sperm binding or fertilization parameters in zona-free oocytes. In conclusion, sperm-zona binding and acrosome reaction were inhibited by serine protease inhibitors during porcine IVF.

  5. Group X phospholipase A2 is released during sperm acrosome reaction and controls fertility outcome in mice

    PubMed Central

    Escoffier, Jessica; Jemel, Ikram; Tanemoto, Akemi; Taketomi, Yoshitaka; Payre, Christine; Coatrieux, Christelle; Sato, Hiroyasu; Yamamoto, Kei; Masuda, Seiko; Pernet-Gallay, Karin; Pierre, Virginie; Hara, Shuntaro; Murakami, Makoto; De Waard, Michel; Lambeau, Gérard; Arnoult, Christophe

    2010-01-01

    Ejaculated mammalian sperm must undergo a maturation process called capacitation before they are able to fertilize an egg. Several studies have suggested a role for members of the secreted phospholipase A2 (sPLA2) family in capacitation, acrosome reaction (AR), and fertilization, but the molecular nature of these enzymes and their specific roles have remained elusive. Here, we have demonstrated that mouse group X sPLA2 (mGX) is the major enzyme present in the acrosome of spermatozoa and that it is released in an active form during capacitation through spontaneous AR. mGX-deficient male mice produced smaller litters than wild-type male siblings when crossed with mGX-deficient females. Further analysis revealed that spermatozoa from mGX-deficient mice exhibited lower rates of spontaneous AR and that this was associated with decreased in vitro fertilization (IVF) efficiency due to a drop in the fertilization potential of the sperm and an increased rate of aborted embryos. Treatment of sperm with sPLA2 inhibitors and antibodies specific for mGX blocked spontaneous AR of wild-type sperm and reduced IVF success. Addition of lysophosphatidylcholine, a catalytic product of mGX, overcame these deficiencies. Finally, recombinant mGX triggered AR and improved IVF outcome. Taken together, our results highlight a paracrine role for mGX during capacitation in which the enzyme primes sperm for efficient fertilization and boosts premature AR of a likely phospholipid-damaged sperm subpopulation to eliminate suboptimal sperm from the pool available for fertilization. PMID:20424324

  6. Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. II. Ultrastructural changes associated with inhibition of the acrosome reaction.

    PubMed

    Chang, M C; Schuel, H

    1991-05-01

    Pretreatment of Strongylocentrotus purpuratus sperm with delta 9-tetrahydrocannabinol (THC) prevents the triggering of the acrosome reaction by egg jelly. Examination of THC-treated sperm by transmission electron microscopy reveals that the membrane fusion reaction between the sperm plasma membrane and the acrosomal membrane is completely blocked. Electron-dense deposits are present in the subacrosomal fossa and in the centriolar fossa. The nuclear envelope is fragmented in close proximity to the electron-dense deposits. The electron-dense deposits are not bound by a limiting membrane, stain positively for lipid with thymol and farnesol, and disappear from THC-treated sperm that are extracted with chloroform:methanol (2:1) after glutaraldehyde fixation. The electron-dense deposits are lipid in nature and may be a hydrolytic product of the nuclear envelope. Electron-dense deposits are seen in sperm after 1-10 min treatment with 5-100 microM THC. The electron-dense deposits disappear after removal of THC from the sperm by washing, but the fragmented nuclear envelope in the subacrosomal fossa persists. Cannabidiol (CBD) and cannabinol (CBN) also inhibit the triggering of the acrosome reaction by egg jelly and produce ultrastructural changes in the sperm identical to those elicited by THC. Enhanced phospholipase activity stimulated by THC, CBD, and CBN may be the cause of the accumulation of lipid deposits in the sperm. Metabolites derived from this modification of membrane phospholipids may prevent triggering of the acrosome reaction by egg jelly and thereby inhibit fertilization.

  7. Identification of egg-jelly substances triggering sperm acrosome reaction in the newt, Cynops pyrrhogaster.

    PubMed

    Watanabe, Akihiko; Fukutomi, Keiko; Kubo, Hideo; Ohta, Manami; Takayama-Watanabe, Eriko; Onitake, Kazuo

    2009-04-01

    Our previous studies have shown that the acrosome reaction (AR) occurs in egg-jelly of the Japanese newt, Cynops pyrrhogaster. This is analogous to the substances of echinoderms but distinct from those of many other vertebrates derived from the egg envelope or its derivative, the zona pellucida. To identify the AR-inducing substances in newt egg jelly, a monoclonal antibody (mAb) was generated against the jelly by screening the culture supernatants to find the one that best neutralized the AR-inducing activity of the jelly substance. The mAb specifically reacted to protein bands in the jelly. These proteins, with apparent molecular weights of 122 and 90 kDa, exhibited AR-inducing activity, indicating that they are definitely AR-inducing substances. Western blotting using the mAb indicated that the 122 and 90 kDa proteins are present only in the egg jelly's outermost layer, where AR-inducing activity is known to occur. Both proteins were recognized with wheat germ agglutinin (WGA), a lectin that inhibits AR-induction in egg jelly extract. Taken together, these findings indicate that the 122 and 90 kDa proteins are the AR-inducing substances in the egg jelly of C. pyrrhogaster. The WGA recognition of the proteins was lost by N-glycosidase digestion, suggesting that N-linked carbohydrate moieties in these proteins may be responsible for the AR-inducing activity.

  8. Phosphorylation of the MAPK pathway has an essential role in the acrosome reaction in miniature pig sperm.

    PubMed

    Kawano, N; Ito, J; Kashiwazaki, N; Yoshida, M

    2010-04-01

    In almost all animal species, sperm acrosome reaction (AR) is a crucial step for fertilization. The step is a Ca(2+)-dependent secretory event that must be completed before fertilization. Many researchers have reported that several chemicals (such as ionomycin, thapsigargin and caffeine) artificially induce this step by increasing [Ca(2+)](i). However, little information has been known on events that occur following Ca(2+) induced initiation of the sperm AR. We show here for the first time that phosphorylation of the mitogen-activated protein kinase (MAPK) pathway is required for the AR in miniature pig sperm. Following caffeine treatment artificially inducing the AR in miniature pig sperm, Raf was phosphorylated and then MAP kinase kinase (MEK) and extracellular-signal regulated kinase 1 (ERK1) were also phosphorylated in a time-dependent manner. However, the total ERK1 level did not change during the culture. Pre-treatment of sperm with U0126, a MEK inhibitor, significantly suppressed both the AR and phosphorylation of MEK/ERK1 in a dose-dependent manner. Additionally, pre-incubation of the sperm with seminal vesicle (SV) fluid, which is known to contain a decapacitation factor, suppressed both the AR and MEK/ERK1 phosphorylation. These results suggest that phosphorylation of MAPK pathway plays an important role in the AR in miniature pig sperm. Moreover, the SV fluid may have an inhibitory effect on the AR via the suppression of the MAPK pathway.

  9. [Values of the sperm deformity index, acrosome abnormity rate, and sperm DNA fragmentation index of optimized sperm in predicting IVF fertilization failure].

    PubMed

    Jiang, Wei-jie; Jin, Fan; Zhou, Li-ming

    2016-02-01

    To investigate the values of the sperm deformity index (SDI), acrosome abnormity rate (AAR), and DNA fragmentation index (DFI) of optimized sperm in the prediction of fertilization failure (fertilization rate < 25%) in conventional in vitro fertilization (IVF). We selected 695 cycles of conventional IVF for pure oviductal infertility in this study, including 603 cycles of normal fertilization and 92 cycles of fertilization failure. On the day of oocyte retrieval, we examined sperm morphology, acrosome morphology, and DNA fragmentation using the Diff-Quik, PSA-FITC and SCD methods. We established the joint predictor (JP) by logistic equation and analyzed the values of different parameters in predicting fertilization failure with the receiver operating characteristic (ROC) curve. The fertilization rate was negatively correlated with SDI (r = - 0.07; P = 0.03), AAR (r = -0.49; P < 0.01), and DFI (r = -0. 21; P < 0.01). The SDI, AAR, and DFI in the normal fertilization group were 1.24 ± 0.20, (7.75 ± 2.28)%, and (7.87 ± 3.15)%, and those in the fertilization failure group were 1.42 ± 0.15, (12.02 ± 3.06)%, and (13.32 ± 4.13)%, respectively, all with statistically significant differences between the two groups (P < 0.05). SDI, AAR, and DFI were all risk factors of fertilization failure ( OR = 2.68, 14.11, and 3.85; P = 0.01, < 0.01, and < 0.01). The areas under the ROC curves for SDI, AAR, DFI, and JP were 0.651 ± 0.033, 0.895 ± 0.019, 0.789 ± 0.022, and 0.915 ± 0.017, respectively. According to the Youden index, the optimal cut-off values of SDI, AAR, and DFI obtained for the prediction of fertilization failure were approximately 1.45, 10%, and 12%. The SDI, AAR and DFI of optimized sperm are closely associated with the fertilization rate, and all have the value for predicting fertilization failure in IVF. The AAR is more valuable than the other single predictors, but JP is more effective than the AAR.

  10. Mechanism of sperm capacitation and the acrosome reaction: role of protein kinases

    PubMed Central

    Ickowicz, Debby; Finkelstein, Maya; Breitbart, Haim

    2012-01-01

    Mammalian sperm must undergo a series of biochemical and physiological modifications, collectively called capacitation, in the female reproductive tract prior to the acrosome reaction (AR). The mechanisms of these modifications are not well characterized though protein kinases were shown to be involved in the regulation of intracellular Ca2+ during both capacitation and the AR. In the present review, we summarize some of the signaling events that are involved in capacitation. During the capacitation process, phosphatidyl-inositol-3-kinase (PI3K) is phosphorylated/activated via a protein kinase A (PKA)-dependent cascade, and downregulated by protein kinase C α (PKCα). PKCα is active at the beginning of capacitation, resulting in PI3K inactivation. During capacitation, PKCα as well as PP1γ2 is degraded by a PKA-dependent mechanism, allowing the activation of PI3K. The activation of PKA during capacitation depends mainly on cyclic adenosine monophosphate (cAMP) produced by the bicarbonate-dependent soluble adenylyl cyclase. This activation of PKA leads to an increase in actin polymerization, an essential process for the development of hyperactivated motility, which is necessary for successful fertilization. Actin polymerization is mediated by PIP2 in two ways: first, PIP2 acts as a cofactor for phospholipase D (PLD) activation, and second, as a molecule that binds and inhibits actin-severing proteins such as gelsolin. Tyrosine phosphorylation of gelsolin during capacitation by Src family kinase (SFK) is also important for its inactivation. Prior to the AR, gelsolin is released from PIP2 and undergoes dephosphorylation/activation, resulting in fast F-actin depolymerization, leading to the AR. PMID:23001443

  11. Potassium ion influx and Na+,K+-ATPase activity are required for the hamster sperm acrosome reaction

    PubMed Central

    1981-01-01

    The role of a K+ ion influx and Na+,K+-ATPase activity in the hamster sperm acrosome reaction (AR) was examined, using a range of concentrations of K+,K+ ionophores and a Na+,K+-ATPase inhibitor. Washed epididymal hamster sperm, capacitated in vitro in an artificial medium containing 2 mM Ca2+, 147 mM Na+, and 3, 6, 12, 18, or 24 mM K+, began undergoing the AR after 3 h of incubation. Sperm incubated in low K+ (0.9 mM) failed to undergo the AR even after 5 h of incubation. Sperm in 0.9 mM K+ could be induced to undergo the AR when either K+ (12 mM) alone or K+ (12 mM) with 0.1 microM nigericin was added after 3.5 h of incubation. The addition of K+ alone stimulated the AR in 30 min, whereas nigericin plus K+ stimulated the AR 15 min after addition. Neither nigericin added alone (0.9 mM K+) nor nigericin plus 12 mM K+ added to a low Ca2+ (0.35 mM) system resulted in acrosome reactions. Valinomycin (1 nM) did not stimulate the AR when added together with K+ (3-24 mM) to sperm incubated in 0.9 mM K+ for 3.5 h but markedly decreased sperm motility. Micromolar levels of ouabain blocked the AR when added between t = 0--3 h to sperm incubated with 3-24 mM K+. Inhibition of AR by the addition of 1 microM ouabain to sperm incubated with 3 mM K+ was completely reversed by the addition of 0.1 microM nigericin at t = 3.5 h. These results suggest that Na+,K+-ATPase activity and the resulting K+ influx are important for the mammalian sperm AR. Some similarities between requirements for the hamster sperm AR and secretory granule exocytosis are discussed. PMID:6271793

  12. Cytochrome c upregulation during capacitation and spontaneous acrosome reaction determines the fate of pig sperm cells: linking proteome analysis.

    PubMed

    Choi, Yun-Jung; Uhm, Sang-Jun; Song, Sang-Jin; Song, Hyuk; Park, Jin-Ki; Kim, Teoan; Park, Chankyu; Kim, Jin-Hoi

    2008-02-01

    To identify the mechanisms underlying capacitation, we undertook a high-resolution differential proteomic analysis of pig sperm cells. Two-dimensional gel electrophoresis and subsequent MALDI-TOF mass spectrometry analyses led to identification of 56 differentially expressed proteins. After induction of capacitation in vitro, the well-established markers of the capacitation (lactadherin P47, acrosomal protein SP-10 precursor, prohibitin, proteasomes, DJ-1 protein and arylsulfatase-A) and TCA cycle proteins (isocitrate dehydrogenase, malate dehydrogenase and pyruvate dehydrogenase) were identified. During induction, cytochrome c expression via the p53 pathway increased, however apoptotic executors, such as caspase-3, decreased significantly. Therefore, we tested the hypothesis that cytochrome c upregulation in spermatozoa is capable of activating tyrosine phosphorylation for capacitation, rather than apoptosis. Exposure of sperm cells to soluble Na2CrO4 [Cr (VI)], which induces cytochrome c upregulation, caused a dose- and time-dependent increase in tyrosine phosphorylation of sperm proteins in non-capacitating medium. In contrast, supplementation of cyclosporin A, which blocks cytochrome c upregulation, inhibited tyrosine phosphorylation of sperm proteins. Furthermore, spermatozoa in capacitation medium or non-capacitation media supplemented with soluble Cr (VI) showed similar levels of capacitation. These findings indicate that differential expression of many of these proteins has previously been unrecognized in sperm cells incubated in capacitation medium also suggest that a gradual increase of cytochrome c during incubation to induce capacitation determines sperm cell fate, i.e., apoptosis or further development for fertilization.

  13. Human sperm acrosome reaction-initiating activity associated with the human cumulus oophorus and mural granulosa cells.

    PubMed

    Siiteri, J E; Dandekar, P; Meizel, S

    1988-04-01

    This report describes the detection and partial characterization of preovulatory human cumulus oophorus and mural granulosa cell-associated activity capable of initiating the human sperm acrosome reaction (AR) in vitro. Fragments of preovulatory human cumulus (cells plus extracellular matrix) were washed 3 times, incubated for 24 hr and the spent media and washes assayed for their ability to initiate the human sperm acrosome reaction (AR) in vitro. AR activity was present in the first two washes but not the third wash; however, AR activity was recovered in the spent medium after 3 X-washed fragments were incubated for 24 hr under conditions which maintained the viability of the cumulus cells. The spent media of preovulatory human mural granulosa cells contained AR-initiating activity after 1-3, 3-6, and 6-9 days of culture. The properties of the AR activity present in spent media of human cumulus fragments included resistance to loss of activity during treatment with pronase; resistance to loss of activity during treatment with chondroitinase ABC or bacterial hyaluronidase; heat stability after overnight incubation; lack of extraction by chloroform-methanol; an apparent molecular weight (MW) of 50,000, as determined by Sephadex G-75 column chromatography; conversion to a lower apparent MW activity by incubation with pronase. These properties are also characteristic of a fraction derived by Sephadex G-75 chromatography of preovulatory human follicular fluid which also has been shown to stimulate the human sperm acrosome reaction in vitro. The AR activity from spent media of human mural granulosa cells is also found in a 50,000 MW Sephadex G-75 fraction. We propose that the sources of the 50,000 MW human follicular fluid AR activity are the cumulus oophorus and the mural granulosa cells.

  14. An intact acrosome is required for the chemotactic response to progesterone in mouse spermatozoa.

    PubMed

    Guidobaldi, Hector A; Hirohashi, Noritaka; Cubilla, Marisa; Buffone, Mariano G; Giojalas, Laura C

    2017-04-01

    Mammalian sperm become fertilization-competent in the oviduct, during a process known as capacitation that involves the acquisition of the ability to exocytose the acrosome but also the chemotactic responses-both of which contribute to successful fertilization. Chemotaxis is used by spermatozoa to orient and to locate the egg; the acrosome reaction facilitates sperm binding to and fusing with the egg membrane. Mammalian spermatozoa are able to sense picomolar concentrations of progesterone, which drives chemotactic behavior. The state of the acrosome during the chemotactic response, however, is unknown. Genetically modified mouse spermatozoa were employed in a chemotaxis assay under fluorescence microscopy to evaluate their acrosome status while swimming, allowing us to elucidate the acrosome integrity of sperm responding to progesterone-induced chemotaxis. We first showed that wild-type mouse spermatozoa chemotactically respond to a gradient of progesterone, and that the genetic modifications employed do not affect the chemotactic behavior of sperm to progesterone. Next, we found that acrosome-intact, but not acrosome-reacted, spermatozoa orient and respond to picomolar concentrations of progesterone and that chemotaxis normally occurs prior to the acrosome reaction. Our results suggest that premature commitment to acrosome exocytosis leads to navigation failure, so proper control and timing of the acrosome reaction is required for fertilization success and male fertility. © 2017 Wiley Periodicals, Inc.

  15. Egg jelly of the newt, Cynops pyrrhogaster contains a factor essential for sperm binding to the vitelline envelope.

    PubMed

    Hiyoshi, Wataru; Sasaki, Takayuki; Takayama-Watanabe, Eriko; Takai, Hiroyuki; Watanabe, Akihiko; Onitake, Kazuo

    2007-06-01

    The acrosome reaction of newt sperm is induced at the surface of egg jelly and the acrosome-reacted sperm acquire the ability to bind to the vitelline envelope. However, because the substance that induces the acrosome reaction has not been identified, the mechanism by which the acrosome-reacted sperm bind to the vitelline envelope remains unclear. We found here that a Dolichos biforus agglutinin (DBA) specifically mimicked the acrosome reaction immediately upon its addition in the presence of milimolar level Ca(2+). Fluorescein isothiocyanate-labeled DBA bound specifically to the acrosomal cap of the intact sperm in the presence of a Ca(2+)-chelating agent, EDTA, suggesting that binding of DBA to the native receptor for the egg jelly substance on the acrosomal region took the place of the egg jelly substance-induced acrosome reaction. In contrast, the sperm that had been acrosome reacted by DBA treatment did not bind to the vitelline envelope of the egg whose jelly layers were removed. Subsequent addition of jelly extract caused the sperm binding to vitelline envelope, indicating that the egg jelly of the newt contains substances that are involved in not only inducing the acrosome reaction but also binding to the vitelline envelope. This is the first demonstration of the involvement of egg jelly substance in the binding of acrosome-reacted sperm to the vitelline envelope.

  16. The Effect of Low-Level Laser Irradiation on Sperm Motility, and Integrity of the Plasma Membrane and Acrosome in Cryopreserved Bovine Sperm

    PubMed Central

    Fernandes, Guilherme Henrique C.; de Carvalho, Paulo de Tarso Camillo; Serra, Andrey Jorge; Crespilho, André Maciel; Peron, Jean Pierre Schatzman; Rossato, Cristiano; Leal-Junior, Ernesto Cesar Pinto; Albertini, Regiane

    2015-01-01

    Background and Objective Freezing changes sperm integrity remarkably. Cryopreservation involves cooling, freezing, and thawing and all these contribute to structural damage in sperm, resulting in reduced fertility potential. Low-level laser irradiation (LLLI) could increase energy supply to the cell and cause reactive oxygen species reduction (ROS), contributing to the restoration of oxygen consumption and adenosine triphosphate synthesis (ATP) in the mitochondria. Our goal was to analyze the effects of low-level laser irradiation on sperm motility and integrity of the plasma membrane and acrosome in cryopreserved bovine sperm. Study Design/Materials and Methods We analyzed 09 samples of bull semen (Bos taurus indicus), divided into three groups: a control group without laser irradiation, a 4J group subjected to a laser irradiation dose of 4 joules, and a 6J group subjected to dose of 6 joules. Samples were divided for the analysis of cell viability and acrosomal membrane integrity using flow cytometry; another portion was used for motion analysis. Irradiation was performed in petri dishes of 30 mm containing 3 ml of semen by an aluminum gallium indium phosphide laser diode with a wavelength of 660 nm, 30 mW power, and energy of 4 and 6 joules for 80 and 120 seconds respectively. Subsequently, the irradiated and control semen samples were subjected to cryopreservation and analyzed by flow cytometry (7AAD and FITC-PSA) using the ISAS - Integrated Semen Analysis System. Results Flow cytometry showed an increase in the percentage of live sperm cells and acrosome integrity in relation to control cells when subjected to irradiation of low-power laser in two different doses of 4 and 6 joules (p < 0.05). In the analysis of straightness, percentage of cell movement, and motility, a dose of 4 joules was more effective (p < 0.05). Conclusion We conclude that LLLI may exert beneficial effects in the preservation of live sperm. A dose of 4 joules prior to cryopreservation was

  17. Molecules that initiate or help stimulate the acrosome reaction by their interaction with the mammalian sperm surface.

    PubMed

    Meizel, S

    1985-11-01

    This review deals with exogenous molecules that stimulate the acrosome reaction (AR) of mammalian sperm in vitro, presumably by acting at the sperm surface. Such molecules may exert their effect(s) by stimulating capacitation and/or by stimulation or initiation of the AR, and they are probably present at one of three putative in vivo sites (also discussed here) for the AR of a fertilizing sperm: the oviductal fluid, the cumulus oophorus matrix, and the zona pellucida. The molecules discussed include serum albumin, hydrolytic enzymes (particularly proteases); hormones including biogenic amines, estradiol, and arachidonic acid metabolites; sulfur-containing beta-amino acids; glycosaminoglycans such as hyaluronic acid; and a zona pellucida glycoprotein. Possible mechanisms to explain the effects of these molecules are also discussed. Several conclusions and suggestions are offered in this review: There is more than one site for the AR of a fertilizing sperm in vitro, depending on experimental conditions and species, but the site(s) at which the AR of a fertilizing sperm occur(s) in vivo is/are still a matter of disagreement; there are a number of molecules that can stimulate or initiate the AR in vitro, and such molecular duplication may also exist in vivo to ensure fertility; and synergistic interaction between some of those exogenous molecules may occur in the stimulation of capacitation and the stimulation or initiation of the AR.

  18. [Application of spontaneous acrosome reaction of sperm in prediction of outcome of in-vitro fertilization and embryo transfer].

    PubMed

    Xuan, X J; Xu, C; Zhao, Y R; Wu, K L; Chen, T; Zhang, H B; Li, X; Su, S Z; Ma, G; Tang, R; Sheng, Y; Ma, J L

    2016-04-26

    To investigate the clinical application of spontaneous acrosome reaction (AR) rate of sperm in predicting the outcome of in-vitro fertilization and embryo transfer (IVF-ET). The spontaneous AR rate of the sperm of patients who underwent IVF-ET treatment in our center during the period from November to December 2014 were studied. The cut-off value from 6% to 12% were set and analyzed its association between the IVF-ET outcomes (including fertility rates, normal fertilization rates and high-quality embryo rates). For those who underwent fresh embryo transplantation, the rates of chemical pregnancy and clinical pregnancy were calculated, and compared the spontaneous AR rates and quantity of acrosomal enzyme according to the pregnancy outcome. There were 202 patients in this study and the mean spontaneous AR rate was 5.99%±5.18%. For patients with the spontaneous AR rate ≥9% versus <9%, the fertility rate, normal fertilization rate and high-quality embryo rate were 81.33% vs 83.85%, 60.53% vs 60.99%, and 51.10% vs 59.67%, respectively, with statistically significant difference in the high-quality embryo rate (P=0.02). For patients who underwent fresh embryo transplantation, when comparison was made between those with spontaneous AR rate ≥8% and those <8%, the rate of chemical pregnancy and clinical pregnancy were 48.57% (17/35) vs 69.64% (78/112) and 37.14% (13/35) vs 63.39% (71/112), respectively, both with statistically significant difference (P=0.02 and P<0.01). The patients with clinical pregnancy had lower spontaneous AR rate than those without clinical pregnancy (5.41%±3.87% vs 7.40%±6.79%, P=0.04), while the quantity of acrosomal enzyme showed no significant difference [(131.79±68.50) vs (153.62±59.59) μU/10(6,) P=0.06]. Logistic regression analysis demonstrated association between spontaneous AR rates and clinical pregnancy (OR=0.93, 95%CI: 0.87-0.99, P=0.03). The spontaneous AR rate of sperm may have clinical significance in predicting the outcome of

  19. Maturational changes in motility, acrosomal proteolytic activity, and penetrability of the inner perivitelline layer of fowl sperm, during their passage through the male genital tract.

    PubMed

    Ahammad, Muslah U; Nishino, C; Tatemoto, H; Okura, N; Kawamoto, Y; Okamoto, S; Nakada, T

    2011-10-01

    The objective was to examine, in vitro, the motility, acrosomal proteolytic activity (APA), and penetrating ability of fowl sperm recovered from the testis and epididymis, as well as the proximal, middle, and distal vas deferens, to assess the potential fertilizing ability of sperm as a function of maturation. A motile sperm separation technique was used to estimate sperm motility with Accudenz, a gelatin slide technique was used to measure the diameter of the halo around the acrosome of individual sperm as an indication of APA, and a sperm-inner perivitelline layer (IPL) interaction assay was done to estimate the number of hole formations as an indication of sperm penetration into the IPL. Sperm in the testis exhibited the least motility, produced the smallest halos, and created the least number of holes per 0.25 mm(2). Motility, diameter of the halo, and number of holes increased gradually (P < 0.05) from the epididymis to the distal vas deferens and were markedly different (P < 0.05) between testicular and deferent duct sperm. Based on these in vitro experimental findings, we inferred that fowl sperm undergo a gradual process of maturational changes in motility, APA, and penetrability as a means of acquiring potential fertility during their passage throughout the male genital tract. Copyright © 2011 Elsevier Inc. All rights reserved.

  20. A Specific Transitory Increase in Intracellular Calcium Induced by Progesterone Promotes Acrosomal Exocytosis in Mouse Sperm1

    PubMed Central

    Romarowski, Ana; Sánchez-Cárdenas, Claudia; Ramírez-Gómez, Héctor V.; Puga Molina, Lis del C.; Treviño, Claudia L.; Hernández-Cruz, Arturo; Darszon, Alberto; Buffone, Mariano G

    2016-01-01

    During capacitation, sperm acquire the ability to undergo the acrosome reaction (AR), an essential step in fertilization. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AR. An increase in intracellular Ca2+ ([Ca2+]i) is necessary for AR to occur. In this study, we investigated the spatiotemporal correlation between the changes in [Ca2+]i and AR in single mouse spermatozoa in response to progesterone. We found that progesterone stimulates an [Ca2+]i increase in five different patterns: gradual increase, oscillatory, late transitory, immediate transitory, and sustained. We also observed that the [Ca2+]i increase promoted by progesterone starts at either the flagellum or the head. We validated the use of FM4-64 as an indicator for the occurrence of the AR by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, we have simultaneously visualized the rise in [Ca2+]i and the process of exocytosis in response to progesterone and found that only a specific transitory increase in [Ca2+]i originating in the sperm head promotes the initiation of AR. PMID:26819478

  1. Involvement of Protein cAMP-dependent Kinase, Phospholipase A2 and Phospholipase C in Sperm Acrosome Reaction of Chinchilla lanigera.

    PubMed

    Gramajo-Bühler, M C; Zelarayán, L; Sánchez-Toranzo, G

    2016-02-01

    The mechanisms involved in fertilization are the centre of attention in order to determine the conditions required to reproduce in vitro the events that take place in vivo, with special interest in endangered species. Previous data from mouse sperm, where acrosome reaction (AR) occurs more often in the interstitium of the cumulus oophorus, contribute to strengthen the use of progesterone as a physiological inducer of this process. We studied the participation of protein kinase A (PKA), phospholipases A2 and C (PLA2 , PLC) in the AR induced by progesterone from Chinchilla epididymal spermatozoa. The addition of db-cAMP to the incubation medium caused an increase of 58% in the AR, while the use of H89 (30 μm), a PKA inhibitor, reflected a decrease of 40% in the percentage of reacted gametes. The assays conducted with arachidonic acid showed a maximum increase of 23% in the AR. When gametes were pre-incubated with PLA2 inhibitors, a dose-dependent inhibitory effect was observed. The addition of phorbol12-myristate13-acetate (10 μm) revealed higher percentages of AR induction (60%). When PLC was inhibited with neomycin and U73122, a dose-dependent decrease in AR percentages was observed. Combined inhibition of PKA, PLA2 and PLC, AR values similar to control were obtained. This work shows evidence, for the first time in Chinchilla, that progesterone activates the AC/cAMP/PKA system as well as sperm phospholipases and that these signalling pathways participate jointly and cooperatively in AR. These results contribute to the understanding of the complex regulation that is triggered in sperm after the effect of progesterone.

  2. Accurate sperm morphology assessment predicts sperm function.

    PubMed

    Abu Hassan Abu, D; Franken, D R; Hoffman, B; Henkel, R

    2012-05-01

    Sperm morphology has been associated with in vitro as well as in vivo fertilisation. The study aimed to evaluate the possible relation between the percentage of spermatozoa with normal morphology and the following sperm functional assays: (i) zona-induced acrosome reaction (ZIAR); (ii) DNA integrity; (iii) chromatin condensation; (iv) sperm apoptosis; and (v) fertilisation rates. Regression analysis was employed to calculate the association between morphology and different functional tests. Normal sperm morphology correlated significantly with the percentages of live acrosome-reacted spermatozoa in the ZIAR (r = 0.518; P < 0.0001; n = 92), DNA integrity (r = -0.515; P = 0.0018; n = 34), CMA(3) -positive spermatozoa (r = -0.745; P < 0.0001; n = 92), sperm apoptosis (r = -0.395; P = 0.0206; n = 34) and necrosis (r = -0.545; P = 0.0009; n = 34). Negative correlations existed between for the acrosome reaction, and DNA integrity, while negative associations were recorded with the percentages of CMA(3) -positive spermatozoa, apoptotic and necrotic spermatozoa. Sperm morphology is related to sperm dysfunction such as poor chromatin condensation, acrosome reaction and DNA integrity. Negative and significant correlations existed between normal sperm morphology and chromatin condensation, the percentage of spermatozoa with abnormal DNA and spermatozoa with apoptotic activity. The authors do not regard sperm morphology as the only test for the diagnosis of male fertility, but sperm morphology can serve as a valuable indicator of underlying dysfunction.

  3. Assessment of sperm hyperactivated motility and acrosome reaction can discriminate the use of spermatozoa for conventional in vitro fertilisation or intracytoplasmic sperm injection: preliminary results.

    PubMed

    Wiser, A; Sachar, S; Ghetler, Y; Shulman, A; Breitbart, H

    2014-04-01

    Basic semen analysis is insufficient for determining the fertility potential. The aim of this study was to determine if hyperactivated motility (HAM) and acrosome reaction (AR) can be useful tests for evaluating semen quality during male infertility evaluations and to help the clinician decide whether regular insemination or intracytoplasmic sperm injection (ICSI) is preferable during in vitro fertilisation. A prospective study was conducted. Patients with normal sperm according to World Health Organization guidelines who underwent IVF treatment and planned regular insemination were asked to participate. A portion of sperm sample was evaluated for HAM and AR on day of ovum pick up. In HAM assessment, 93.3% of patients with increased HAM had a high fertilisation rate compared with 64% in the group without increased HAM (P = 0.059). For the AR evaluation, 91.7% of samples with a low rate of spontaneous AR had a high fertilisation rate compared with 39.3% in the group with a high rate of spontaneous AR (P = 0.004).

  4. Membrane events in the acrosomal reaction of Limulus sperm. Membrane fusion, filament-membrane particle attachment, and the source and formation of new membrane surface

    PubMed Central

    1979-01-01

    The membranes of Limulus (horseshoe crab) sperm were examined before and during the acrosomal reaction by using the technique of freeze- fracturing and thin sectioning. We focused on three areas. First, we examined stages in the fusion of the acrosomal vacuole with the cell surface. Fusion takes place in a particle-free zone which is surrounded by a circlet of particles on the P face of the plasma membrane and an underlying circlet of particles on the P face of the acrosomal vauole membrane. These circlets of particles are present before induction. Up to nine focal points of fusion occur within the particle-free zone. Second, we describe a system of fine filaments, each 30 A in diameter, which lies between the acrosomal vacuole and the plasma membrane. These filaments change their orientation as the vacuole opens, a process that takes place in less than 50 ms. Membrane particles seen on the P face of the acrosomal vacuole membrane change their orientation at the same time and in the same way as do the filaments, thus indicating that the membrane particles and filaments are probably connected. Third, we examined the source and the point of fusion of new membrane needed to cover the acrosomal process. This new membrane is almost certainly derived from the outer nuclear envelope and appears to insert into the plasma membrane in a particle-free area adjacent to an area rich in particles. The latter is the region where the particles are probably connected to the cytoplasmic filaments. The relevance of these observations in relation to the process of fertilization of this fantastic sperm is discussed. PMID:582596

  5. Correlation analysis of the progesterone-induced sperm acrosome reaction rate and the fertilisation rate in vitro.

    PubMed

    Jiang, T; Qin, Y; Ye, T; Wang, Y; Pan, J; Zhu, Y; Duan, L; Li, K; Teng, X

    2015-10-01

    In this study, we aimed to investigate whether progesterone-induced acrosome reaction (AR) rate could be an indicator for fertilisation rate in vitro. Twenty-six couples with unexplained infertility and undergoing in vitro fertilisation (IVF) treatment were involved. On the oocytes retrieval day after routine IVF, residual sperm samples were collected to receive progesterone induction (progesterone group) or not (control group). AR rate was calculated and fertilisation rate was recorded. The correlation between progesterone-induced AR and fertilisation rate and between sperm normal morphology and 3PN (tripronuclear) were analysed using the Spearman correlation analysis. The AR rate of progesterone group was statistically higher than that of the control group (15.6 ± 5.88% versus 9.66 ± 5.771%, P < 0.05), but not significantly correlated with fertilisation rate (r = -0.053, P > 0.01) or rate of high-quality embryo development (r = -0.055, P > 0.01). Normal sperm morphology also showed no significant correlation with the amount of 3PN zygotes (r = 0.029, P > 0.01), rate of 3PN zygotes production (r = 0.20, P > 0.01), rate of 3PN embryo development (r = -0.406, P > 0.01), fertilisation rate (r = -0.148, P > 0.01) or progesterone-induced AR rate (r = 0.214, P > 0.01). Progesterone can induce AR in vitro significantly; however, the progesterone-induced AR may not be used to indicate fertilisation rate.

  6. Fucose, mannose, and β-N-acetylglucosamine glycopolymers initiate the mouse sperm acrosome reaction through convergent signaling pathways.

    PubMed

    Wu, Linghui; Sampson, Nicole S

    2014-02-21

    The sperm acrosome reaction (AR), an essential exocytosis step in mammalian fertilization, is mediated by a species-specific interaction of sperm surface molecules with glycans on the egg. Previous studies indicate that a subset of terminal carbohydrates on the mouse egg zona pellucida (ZP) trigger the AR by cross-linking or aggregating receptors on the sperm membrane. However, the exact role of those carbohydrates in AR has not been identified and the mechanism underlying the AR still needs further investigation. To study this process, a series of glycopolymers was synthesized. The glycopolymers are composed of a multivalent scaffold (norbornene), a functional ligand (previously identified ZP terminal monosaccharides), and a linker connecting the ligand and the scaffold. The polymers were tested for their ability to initiate AR and through which signaling pathways AR induction occurred. Our data demonstrate that mannose, fucose, and β-N-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent manner, and the 100-mers are more potent on a per monomer basis than the 10-mers. Although nearly equipotent in inducing the AR at the optimal concentrations, their AR activation kinetics are not identical. Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding regulatory proteins (G-proteins), tyrosine kinase, protein kinase A, protein kinase C, and Ca(2+)-related antagonists. Thus, the chemotypes of synthetic glycopolymers imitate the physiologic AR-activation agents and provide evidence that occupation of one of at least three different receptor binding sites is sufficient to initiate the AR.

  7. Motility, acrosome integrity, membrane integrity and oocyte cleavage rate of sperm separated by swim-up or Percoll gradient method from frozen-thawed buffalo semen.

    PubMed

    Mehmood, A; Anwar, M; Naqvi, S M Saqlan

    2009-04-01

    Frozen-thawed semen of five buffalo bulls was used to compare efficacy of swim-up and Percoll gradient methods for separating viable spermatozoa. Sperm separated by the two methods were also tested to differentiate buffalo bulls on the basis of in vitro fertilization (IVF) rates. Recovery of motile sperm (%), increase in membrane integrity (%) and acrosome integrity (%) were compared after two sperm separation methods in experiment I, and in vitro fertilization rate (cleavage rate and cleavage index) was compared in experiment II. Swim-up separated sperm showed a higher motility (P<0.05), while percent recovery of motile sperm was higher with Percoll separation (P<0.05). Membrane integrity (%) of sperm separated with swim-up was significantly higher (P<0.05) as compared to sperm separated with Percoll gradient. Swim-up separated sperm gave a higher cleavage rate and cleavage index (P<0.001). Sperm separated by swim-up showed significant difference among the bulls in cleavage rate and cleavage index (P<0.05), while the Percoll gradient method did not. It has been concluded that separation of sperm from frozen-thawed buffalo semen by swim-up method can be more expedient for IVF in buffalo.

  8. Sequence and domain organization of scruin, an actin-cross-linking protein in the acrosomal process of Limulus sperm

    PubMed Central

    1995-01-01

    The acrosomal process of Limulus sperm is an 80-microns long finger of membrane supported by a crystalline bundle of actin filaments. The filaments in this bundle are crosslinked by a 102-kD protein, scruin present in a 1:1 molar ratio with actin. Recent image reconstruction of scruin decorated actin filaments at 13-A resolution shows that scruin is organized into two equally sized domains bound to separate actin subunits in the same filament. We have cloned and sequenced the gene for scruin from a Limulus testes cDNA library. The deduced amino acid sequence of scruin reflects the domain organization of scruin: it consists of a tandem pair of homologous domains joined by a linker region. The domain organization of scruin is confirmed by limited proteolysis of the purified acrosomal process. Three different proteases cleave the native protein in a 5-kD Protease-sensitive region in the middle of the molecule to generate an NH2-terminal 47-kD and a COOH-terminal 56-kD protease-resistant domains. Although the protein sequence of scruin has no homology to any known actin-binding protein, it has similarities to several proteins, including four open reading frames of unknown function in poxviruses, as well as kelch, a Drosophila protein localized to actin-rich ring canals. All proteins that show homologies to scruin are characterized by the presence of an approximately 50-amino acid residue motif that is repeated between two and seven times. Crystallographic studies reveal this motif represents a four beta-stranded fold that is characteristic of the "superbarrel" structural fold found in the sialidase family of proteins. These results suggest that the two domains of scruin seen in EM reconstructions are superbarrel folds, and they present the possibility that other members of this family may also bind actin. PMID:7822422

  9. In vitro effects of l-carnitine and glutamine on motility, acrosomal abnormality, and plasma membrane integrity of rabbit sperm during liquid-storage.

    PubMed

    Sarıözkan, Serpil; Ozdamar, Saim; Türk, Gaffari; Cantürk, Fazile; Yay, Arzu

    2014-06-01

    This study was designed to evaluate the in vitro effects of l-carnitine and glutamine (Gln) on the sperm quality parameters of liquid-stored rabbit semen maintained up to 24 h at 5°C. Pooled and extended ejaculates were divided into two equal portions. l-Carnitine doses of 0.5, 1 and 2mM were added to the first portion, and glutamine was added at the same doses to the second portion. All samples were cooled to 5°C and examined at 0, 6, 12 and 24 h of liquid storage. Supplementation of the semen extender with three different doses of l-carnitine provided significant increases in the percentage of motile sperm at 12 h (P<0.01), and 24h (P<0.001) and enabled significant protection of the sperm plasma membrane (P<0.01) at 12 and 24h of cool-storage, in comparison to the control samples. Only the 2mM dose of l-carnitine significantly (P<0.01) decreased the rate of acrosomal damage when compared to the control group. Furthermore, all doses of Gln caused a significant (P<0.01) decrease in acrosomal damage at 6h, and provided significant improvement (P<0.01) in sperm motility, acrosomal and plasma membrane integrities at 12 and 24h of liquid storage, when compared to the controls. In conclusion, the supplementation of liquid-stored rabbit semen with l-carnitine and Gln provided a protection for sperm against cool storage-induced functional and structural damages.

  10. Slow cooling prevents cold-induced damage to sperm motility and acrosomal integrity in the black-footed ferret (Mustela nigripes).

    PubMed

    Santymire, R M; Marinari, P E; Kreeger, J S; Wildt, D E; Howard, J G

    2007-01-01

    The endangered black-footed ferret (Mustela nigripes) has benefited from artificial insemination; however, improved sperm cryopreservation protocols are still needed. The present study focused on identifying factors influencing gamete survival during processing before cryopreservation, including: (1) the presence or absence of seminal plasma; (2) temperature (25 degrees C v. 37 degrees C); (3) type of medium (Ham's F10 medium v. TEST yolk buffer [TYB]); (4) cooling rate (slow, rapid and ultra-rapid); and (5) the presence or absence of glycerol. Seminal plasma did not compromise (P > 0.05) sperm motility or acrosomal integrity. Sperm motility traits were maintained longer (P < 0.05) at 25 degrees C than at 37 degrees C in Ham's or TYB, but temperature did not affect (P > 0.05) acrosomal integrity. Overall, TYB maintained optimal (P < 0.05) sperm motility compared with Ham's medium, but Ham's medium maintained more (P < 0.05) intact acrosomes than TYB. Slow cooling (0.2 degrees C min(-1)) was optimal (P < 0.05) compared to rapid cooling (1 degrees C min(-1)), and ultra-rapid cooling (9 degrees C min(-1)) was found to be highly detrimental (P < 0.05). Results obtained in TYB with 0% or 4% glycerol were comparable (P > 0.05), indicating that 4% glycerol was non-toxic to ferret sperm; however, glycerol failed to ameliorate the detrimental effects of either rapid or ultra-rapid cooling. The results of the present study demonstrate that the damage observed to black-footed ferret spermatozoa is derived largely from the rate of cooling.

  11. Molecular Cloning of Spergen-4, Encoding a Spermatogenic Cell-Specific Protein Associated with Sperm Flagella and the Acrosome Region in Rat Spermatozoa.

    PubMed

    Howida, Ali; Salaheldeen, Elsaid; Iida, Hiroshi

    2016-04-01

    We used a differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene LOC690919 with an open reading frame of 1227-length nucleotides encoding a protein of 409 amino acids. This gene was designated as Spergen-4 (a spermatogenic cell-specific gene-4). Spergen-4 mRNA was highly expressed in testis, and its expression was detected in rat testis starting at three weeks of postnatal development and persisting up to adulthood. Mouse and human orthologs, which lack N-terminal 77 amino acid residues of rat Spegen-4, were found in the database. Immunofluorescence microscopy and immunoblot analysis demonstrated that Spergen-4 was not expressed in spermatogonia, spermatocytes, and round spermatids, but was restrictedly detected at sperm head, cytoplasm, and developing flagella of elongated spermatids in rat testis. In mature spermatozoa, Spergen-4 was detected at the acrosome region as well as the principal piece of flagella. Spergen-4 immunosignal disappeared from sperm heads on acrosome reaction induced by progesterone. These data suggest that Spergen-4 integrated into elongated spermatids during spermiogenesis serves as a constituent for acrosome region and flagella of rat spermatozoa.

  12. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form.

    PubMed

    Zheng, H; Mandal, A; Shumilin, I A; Chordia, M D; Panneerdoss, S; Herr, J C; Minor, W

    2015-07-01

    Sperm lysozyme-like protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15 Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75 Å in diameter with a 25 Å central pore comprised of six monomers per helix turn repeating every 33 Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan-binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan-binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally observed SLLP1/SAS1B interaction involved in fertilization. © 2015 American Society of Andrology and European Academy of Andrology.

  13. Sperm Lysozyme-Like Protein 1 (SLLP1), an intra-acrosomal oolemmal-binding sperm protein, reveals filamentous organization in protein crystal form

    PubMed Central

    Zheng, Heping; Mandal, Arabinda; Shumilin, Igor A.; Chordia, Mahendra D.; Panneerdoss, Subbarayalu; Herr, John C.; Minor, Wladek

    2016-01-01

    Sperm Lysozyme-Like Protein 1 (SLLP1) is one of the lysozyme-like proteins predominantly expressed in mammalian testes that lacks bacteriolytic activity, localizes in the sperm acrosome, and exhibits high affinity for an oolemmal receptor, SAS1B. The crystal structure of mouse SLLP1 (mSLLP1) was determined at 2.15Å resolution. mSLLP1 monomer adopts a structural fold similar to that of chicken/mouse lysozymes retaining all four canonical disulfide bonds. mSLLP1 is distinct from c-lysozyme by substituting two essential catalytic residues (E35T/D52N), exhibiting different surface charge distribution, and by forming helical filaments approximately 75Å in diameter with a 25Å central pore comprised of six monomers per helix turn repeating every 33Å. Cross-species alignment of all reported SLLP1 sequences revealed a set of invariant surface regions comprising a characteristic fingerprint uniquely identifying SLLP1 from other c-lysozyme family members. The fingerprint surface regions reside around the lips of the putative glycan binding groove including three polar residues (Y33/E46/H113). A flexible salt bridge (E46-R61) was observed covering the glycan binding groove. The conservation of these regions may be linked to their involvement in oolemmal protein binding. Interaction between SLLP1 monomer and its oolemmal receptor SAS1B was modeled using protein-protein docking algorithms, utilizing the SLLP1 fingerprint regions along with the SAS1B conserved surface regions. This computational model revealed complementarity between the conserved SLLP1/SAS1B interacting surfaces supporting the experimentally-observed SLLP1/SAS1B interaction involved in fertilization. PMID:26198801

  14. Sperm gamma-aminobutyric acid type A receptor delta subunit (GABRD) and its interaction with purinergic P2X2 receptors in progesterone-induced acrosome reaction and male fertility.

    PubMed

    Xu, Wenming; Wang, Ke; Chen, Yan; Liang, Xiao Tong; Yu, Mei Kuen; Yue, Huanxun; Tierney, M Louise

    2017-02-13

    The mechanism underlying the non-genomic action of progesterone in sperm functions and related Ca2+ mobilisation remains elusive. Herein we report the expression of gamma-aminobutyric acid type A receptor delta subunit (GABRD) in human and rodent sperm and its involvement in mediating the progesterone-induced acrosome reaction. GABRD was localised in the sperm head/neck region. A δ(392-422)-specific inhibitory peptide against GABRD blocked the progesterone-induced acrosome reaction and the associated increase in intracellular Ca2+. Similarly, an inhibitory effect against both progesterone-induced Ca2+ influx and the acrosome reaction was observed with a P2X2 receptor antagonist. The lack of synergism between the GABRD and P2X2 inhibitors suggests that these two receptors are playing a role in the same pathway. Furthermore, a co-immunoprecipitation experiment demonstrated that GABRD could undergo protein-protein interactions with the Ca2+-conducting P2X2 receptor. This interaction between the receptors could be reduced following progesterone (10μM) inducement. Significantly reduced GABRD expression was observed in spermatozoa from infertile patients with reduced acrosome reaction capacity, suggesting that normal expression of GABRD is critical for the sperm acrosome reaction and thus male fertility. The results of the present study indicate that GABRD represents a novel progesterone receptor or modulator in spermatozoa that is responsible for the progesterone-induced Ca2+ influx required for the acrosome reaction through its interaction with the P2X2 receptor.

  15. Sperm whales (Physeter catodon L. 1758) do not react to sounds from detonators.

    PubMed

    Madsen, P T; Møhl, B

    2000-01-01

    A number of observations show that sperm whales (Physeter catodon L. 1758) react to various man-made pulses with moderate source levels. The behavioral responses are described to vary from silence to fear. Click rates of five submerged male sperm whales were measured during the discharge of eight detonators off Andenes, northern Norway. In addition, the behavioral response of a surfaced specimen was observed. Click rates of the submerged whales and the behavior of the surfaced specimen did not change during the discharges with received sound levels of some 180 dB re 1 microPa peRMS. The apparent lack of response to the discharges could be due to similarity between sperm whale clicks and detonations. Accordingly, it can be speculated that the discharges may have been perceived as isolated clicks from conspecifics.

  16. A Recurrent Deletion of DPY19L2 Causes Infertility in Man by Blocking Sperm Head Elongation and Acrosome Formation

    PubMed Central

    Harbuz, Radu; Zouari, Raoudha; Pierre, Virginie; Ben Khelifa, Mariem; Kharouf, Mahmoud; Coutton, Charles; Merdassi, Ghaya; Abada, Farid; Escoffier, Jessica; Nikas, Yorgos; Vialard, François; Koscinski, Isabelle; Triki, Chema; Sermondade, Nathalie; Schweitzer, Thérèse; Zhioua, Amel; Zhioua, Fethi; Latrous, Habib; Halouani, Lazhar; Ouafi, Marrakchi; Makni, Mounir; Jouk, Pierre-Simon; Sèle, Bernard; Hennebicq, Sylviane; Satre, Véronique; Viville, Stéphane; Arnoult, Christophe; Lunardi, Joël; Ray, Pierre F.

    2011-01-01

    An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization. PMID:21397064

  17. A recurrent deletion of DPY19L2 causes infertility in man by blocking sperm head elongation and acrosome formation.

    PubMed

    Harbuz, Radu; Zouari, Raoudha; Pierre, Virginie; Ben Khelifa, Mariem; Kharouf, Mahmoud; Coutton, Charles; Merdassi, Ghaya; Abada, Farid; Escoffier, Jessica; Nikas, Yorgos; Vialard, François; Koscinski, Isabelle; Triki, Chema; Sermondade, Nathalie; Schweitzer, Thérèse; Zhioua, Amel; Zhioua, Fethi; Latrous, Habib; Halouani, Lazhar; Ouafi, Marrakchi; Makni, Mounir; Jouk, Pierre-Simon; Sèle, Bernard; Hennebicq, Sylviane; Satre, Véronique; Viville, Stéphane; Arnoult, Christophe; Lunardi, Joël; Ray, Pierre F

    2011-03-11

    An increasing number of couples require medical assistance to achieve a pregnancy, and more than 2% of the births in Western countries now result from assisted reproductive technologies. To identify genetic variants responsible for male infertility, we performed a whole-genome SNP scan on patients presenting with total globozoospermia, a primary infertility phenotype characterized by the presence of 100% round acrosomeless spermatozoa in the ejaculate. This strategy allowed us to identify in most patients (15/20) a 200 kb homozygous deletion encompassing only DPY19L2, which is highly expressed in the testis. Although there was no known function for DPY19L2 in humans, previous work indicated that its ortholog in C. elegans is involved in cell polarity. In man, the DPY19L2 region has been described as a copy-number variant (CNV) found to be duplicated and heterozygously deleted in healthy individuals. We show here that the breakpoints of the deletions are located on a highly homologous 28 kb low copy repeat (LCR) sequence present on each side of DPY19L2, indicating that the identified deletions were probably produced by nonallelic homologous recombination (NAHR) between these two regions. We demonstrate that patients with globozoospermia have a homozygous deletion of DPY19L2, thus indicating that DPY19L2 is necessary in men for sperm head elongation and acrosome formation. A molecular diagnosis can now be proposed to affected men; the presence of the deletion confirms the diagnosis of globozoospermia and assigns a poor prognosis for the success of in vitro fertilization.

  18. Sulfated polysaccharides from the egg jelly layer are species-specific inducers of acrosomal reaction in sperms of sea urchins.

    PubMed

    Alves, A P; Mulloy, B; Diniz, J A; Mourão, P A

    1997-03-14

    We have characterized the fine structure of sulfated polysaccharides from the egg jelly layer of three species of sea urchins and tested the ability of these purified polysaccharides to induce the acrosome reaction in spermatozoa. The sea urchin Echinometra lucunter contains a homopolymer of 2-sulfated, 3-linked alpha-L-galactan. The species Arbacia lixula and Lytechinus variegatus contain linear sulfated alpha-L-fucans with regular tetrasaccharide repeating units. Each of these sulfated polysaccharides induces the acrosome reaction in conspecific but not in heterospecific spermatozoa. These results demonstrate that species specificity of fertilization in sea urchins depends in part on the fine structure of egg jelly sulfated polysaccharide.

  19. Effects of cryopreservation on the acrosomal status of stallion spermatozoa.

    PubMed

    Bedford, S J; Varner, D D; Meyers, S A

    2000-01-01

    The effects of cryopreservation on the acrosomal status of equine spermatozoa were investigated. Ejaculates (n=10) from six stallions were processed fresh, after cooled storage at 4-6 degrees C for 24 h in either a milk-based or lactose-EDTA freezing extender and after freeze-thawing in lactose-EDTA extender in liquid nitrogen at either 5 x 10(7) or 2 x 10(8) spermatozoa ml(-1). All samples were incubated in TALP-TEST for 2 h at 39 degrees C in 5% CO2. Subsamples were challenged with calcium ionophore A23187 for 10 min. The acrosomal status of the spermatozoa was evaluated by staining the spermatozoa with FITC-conjugated Pisum sativum agglutinin, with ethidium homodimer as a nuclear counterstain. Sperm cell viability was assessed with Hoechst 33258. The data were analysed by a general linear model ANOVA (P < 0.05). Treatment with calcium ionophore did not affect the percentage of acrosome reactions. The samples containing 5 x 10(7) and 2 x 10(8) spermatozoa ml(-1) that were frozen in lactose-EDTA extender in liquid nitrogen had higher percentages of spermatozoa in intermediate categories of acrosomal staining than did any other treatment. The percentage of acrosome-reacted cells was also higher overall for these samples. The percentage of viable cells was lowest for the sperm samples frozen in lactose-EDTA extender, and lower in semen stored in either a milk-based or lactose-EDTA freezing extender than in fresh semen. In conclusion, freeze-thawing resulted in a high percentage of acrosomal changes and a significant decrease in sperm viability.

  20. The activity for the induction of the sperm acrosome reaction localises in the outer layers and exists in the high-molecular-weight components of the egg-jelly of the newt, Cynops pyrrhogaster.

    PubMed

    Sasaki, Takayuki; Kamimura, Saori; Takai, Hiroyuki; Watanabe, Akihiko; Onitake, Kazuo

    2002-02-01

    Localisation of the acrosome reaction inducing activity in egg-jelly was examined in the newt, Cynops pyrrhogaster. The jelly has six layers: the J0, J1, J2, J3, J4 and st layers. Jelly was mechanically dissected and placed on a Millipore filter. When sperm were added from the outer surface side of the jelly, most of them exhibited the acrosome reaction after passing through the jelly. When egg-jelly was divided into four layers, strong activity for the induction of acrosome reaction was detected in the outer layers, J4+st. These findings suggest that the acrosome reaction is induced by a substance in the outer layers of the egg-jelly. Among jelly components separated by SDS-PAGE, a fraction of more than 500 kDa in molecular weight induced the acrosome reaction. Wheat germ agglutinin (WGA), Griffonia simplicifoliar agglutinin 1 (GS-1), Maclura pomifera agglutinin (MPA) and Arachis hypogaea agglutinin (PNA) inhibited the induction of the acrosome reaction by jelly extract, and WGA did so in a dose-dependent manner. Those lectins precipitated some molecules of over 500 kDa. These results suggest that the acrosome reaction is induced by the high molecular-weight components of egg-jelly in C. pyrrhogaster.

  1. The opioid peptide beta-endorphin stimulates acrosome reaction in human spermatozoa.

    PubMed

    Urizar-Arenaza, I; Estomba, H; Muñoa-Hoyos, I; Matorras, R; Esposito, A; Candenas, L; Pinto, F M; Valdivia, A; Irazusta, J; Subirán, N

    2016-01-01

    The acrosome reaction occurs in vivo following sperm capacitation and is essential for the acquisition of sperm fertilization ability. However, little is known about the molecular identity of the physiological acrosome reaction regulators. In addition to progesterone, which is produced by cumulus oophorus cells and known to regulate acrosome reaction by activating the specific calcium channel CatSper, endogenous opioid peptides such as beta-endorphin and met-enkephalin are present at high concentrations in the follicular fluid suggesting that the opioid system may be involved in the mechanisms regulating the acrosome reaction in humans. By using Reverse Transcription-PCR, western blot and immunofluorescence approaches, we described the presence and localization of the beta-endorphin precursor, pro-opiomelanocortinin the middle section and in flagellum of human spermatozoa, and inside the seminiferous tubules of human testis. Flow cytometry and intracellular calcium analyses showed that beta-endorphin causes an inversely dose-dependent increase in the percentage of acrosome-reacted sperm cells by a calcium-independent protein kinase C pathway. These findings are important for future studies of sperm physiology and provide new insight into the function of the opioid system as a target of fertility management.

  2. Acrosomal and viability status of bovine spermatozoa evaluated by two staining methods.

    PubMed

    Jankovicová, Jana; Simon, Michal; Antalíková, Jana; Horovská, L'ubica

    2008-03-01

    Artificial insemination with frozen-thawed spermatozoa is commonly used in cattle breeding. A simple and fast procedure is needed for routine evaluation of the acrosomal status of frozen-thawed bovine sperm. Therefore, the purpose of this study was to test two staining procedures used to determine the viability and integrity of acrosome of frozen-thawed bovine spermatozoa. Double staining and Hoechst/FITC-Pisum sativum agglutinin (FITC-PSA) labelling were tested for evaluating the viability and acrosome reaction induced by calcium ionophore of bull spermatozoa. In our experiments no significant differences were detected in the frequency of acrosome-reacted sperm either by double staining (37.98%) or by FITC-PSA labelling (39.33%). The viability of sperm stained by the double staining method was 67.17%, and a higher portion of viable sperm (82.67%) was observed by staining with the Hoechst procedure (P < 0.01). On the basis of the results obtained it is concluded that both methods can be used for detecting the acrosome reaction of frozen-thawed bovine spermatozoa.

  3. Kinetics of onset of mouse sperm acrosome reaction induced by solubilized zona pellucida: fluorimetric determination of loss of pH gradient between acrosomal lumen and medium monitored by dapoxyl (2-aminoethyl) sulfonamide and of intracellular Ca(2+) changes monitored by fluo-3.

    PubMed

    Rockwell, P L; Storey, B T

    2000-03-01

    The onset of the zona pellucida-induced acrosome reaction in mouse sperm is marked by loss of the pH gradient existing in acrosome-intact sperm between the acidic acrosomal lumen and the suspending medium, due to pore formation between outer acrosomal and plasma membranes. In earlier work, it was shown that this pH gradient loss occurred in single sperm bound to structurally intact zonae pellucidae with a half-time of 2.1 min; the extended kinetics of this loss determined in a sperm population bound to intact zonae was due to a 180-min range of variable lag times. We hypothesized that this lag time range was due to steric constraints imposed by the three-dimensional structure of the structurally intact zona pellucida, and that this constraint should be removed in solubilized zonae. The fluorescent probe, Dapoxyl(TM) (2-aminoethyl)sulfonamide (DAES) allowed a test of this hypothesis in a population of sperm cells. It is a weak base that is non-fluorescent in aqueous solution, but which accumulates in the acidic acrosomal compartment due to the pH gradient with highly enhanced fluorescence; loss of the pH gradient leads to a decrease in fluorescence. The half-time for DAES fluorescence loss in a population of capacitated, acrosome-intact sperm in response to solubilized zona pellucida protein was 2.13 +/- 0.10 min (SEM, n = 9). The agreement between single cell and cell population kinetics validates the hypothesis of steric constraint in the structurally intact zona pellucida. The change in intracellular Ca(2+) concentration in response to solubilized zona pellucida, as monitored with fluo-3, was a rapid increase, followed by a decrease, with a half-time of 0.85 +/- 0.09 min (SEM, n = 6) to a steady state level higher than the initial level, indicating this Ca(2+) transient as the precursor reaction to onset of the zona-induced acrosome reaction. Copyright 2000 Wiley-Liss, Inc.

  4. Expression of the male reproduction-related gene in spermatic ducts of the blue swimming crab, Portunus pelagicus, and transfer of modified protein to the sperm acrosome.

    PubMed

    Sroyraya, Morakot; Hanna, Peter J; Changklungmoa, Narin; Senarai, Thanyaporn; Siangcham, Tanapan; Tinikul, Yotsawan; Sobhon, Prasert

    2013-01-01

    Expression of a sex-specific gene in Macrobrachium rosenbergii (Mr-Mrr), encoding a male reproduction-related (Mrr) protein, has been identified in the spermatic ducts (SDs) and postulated to be involved in sperm maturation processes. M. rosenbergii is the only decapod that the expression and fate of the Mrr protein has been studied. To determine that this protein was conserved in decapods, we firstly used cloning techniques to identify the Mrr gene in two crabs, Portunus pelagicus (Pp-Mrr) and Scylla serrata (Ss-Mrr). We then investigated expression of Pp-Mrr by in situ hybridization, and immunolocalization, as well as phosphorylation and glycosylation modifications, and the fate of the protein in the male reproductive tract. Pp-Mrr was shown to have 632 nucleotides, and a deduced protein of 110 amino acids, with an unmodified molecular weight of 11.79 kDa and a mature protein with molecular weight of 9.16 kDa. In situ hybridization showed that Pp-Mrr is expressed in the epithelium of the proximal, middle, distal SDs, and ejaculatory ducts. In Western blotting, proteins of 10.9 and 17.2 kDa from SDs were all positive using anti-Mrr, antiphosphoserine/threonine, and antiphosphotyrosine. PAS staining showed they were also glycosylated. Immunolocalization studies showed Pp-Mrr in the SD epithelium, lumen, and on the acrosomes of spermatozoa. Immunofluorescence staining indicated the acrosome of spermatozoa contained the Mrr protein, which is phosphorylated with serine/threonine and tyrosine, and also glycosylated. The Mrr is likely to be involved in acrosomal activation during fertilization of eggs.

  5. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes.

    PubMed

    Anzar, M; Kroetsch, T; Boswall, L

    2011-06-01

    In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.

  6. Recombinant human ZP3-induced sperm acrosome reaction: evidence for the involvement of T- and L-type voltage-gated calcium channels.

    PubMed

    José, Omar; Hernández-Hernández, Oscar; Chirinos, Mayel; González-González, María Elena; Larrea, Fernando; Almanza, Angélica; Felix, Ricardo; Darszon, Alberto; Treviño, Claudia L

    2010-05-14

    For successful fertilization mammalian spermatozoa must undergo the acrosome reaction (AR), an exocytotic event that allows this cell to penetrate the outer layer of the oocyte, the zona pellucida (ZP). Four glycoproteins (ZP1-ZP4) compose the human ZP, being ZP3 the physiological inductor of the AR. This process requires changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) involving not fully understood mechanisms. Even in mouse sperm, the pharmacologically documented participation of voltage-gated Ca(2+) (Ca(V)) channels and store-operated channels (SOCs) in the ZP-induced AR is being debated. The situation in human sperm is even less clear due to the limited availability of human ZP. Here, we used recombinant human ZP3 (rhZP3) produced in baculovirus-infected Sf9 cells to investigate the involvement of Ca(V) channels in the human sperm AR. Our findings showed that Ni(2+) and mibefradil at concentrations that block T-type or Ca(V)3 channels, and nimodipine and diltiazem that block L-type or Ca(V)1 channels, significantly inhibited the rhZP3-initiated AR. On the other hand, the AR was insensitive to concentrations of omega-Agatoxin IVA, omega-Conotoxin GVIA and SNX-482 that block P/Q, N and R-type channels, respectively (Ca(V)2 channels). Our overall findings suggest that Ca(V)1 and Ca(V)3 channels participate in human sperm AR. Consistent with this, we detected in human sperm transcripts for the Ca(V)1 auxiliary subunits, alpha(2)delta, beta(1), beta(2) and beta(4), but not the neuronal specific isoforms beta(3) and gamma(2).

  7. Changes of IZUMO1 in bull spermatozoa during the maturation, acrosome reaction, and cryopreservation.

    PubMed

    Fukuda, Masaki; Sakase, Mitsuhiro; Fukushima, Moriyuki; Harayama, Hiroshi

    2016-12-01

    To obtain basic information of bull IZUMO1 (a sperm protein essential for sperm-egg fusion) and disclose possible causes for the impaired fertilizing ability in bull cryopreserved spermatozoa, we investigated this protein in bull spermatozoa collected from various regions of epididymides, freshly ejaculated spermatozoa, acrosome-reacted spermatozoa, and cryopreserved spermatozoa by Western blotting and the triple staining with the anti-IZUMO1 antibody, fluorescein isothiocyanate-peanut agglutinin, and 4',6-diamidino-2-phenylindole. In the cauda epididymal spermatozoa and freshly ejaculated spermatozoa, bull IZUMO1 was detected mainly as a 45-kDa major form. This major form was derived probably from a 52-kDa precursor form in the epididymis. Bull IZUMO1 was immunolocalized along the border between the principal and equatorial segments of the acrosomal region (pattern P1 of IZUMO1) in the most of epididymal and freshly ejaculated spermatozoa with normal acrosomes. In the samples after the treatments to induce the acrosome reaction, the percentages of spermatozoa without acrosomes and with IZUMO1 in whole equatorial segment (pattern P2 of IZUMO1) significantly increased. These results indicate that bull IZUMO1 undergoes maturation-related changes during sperm transit through the epididymis and that it is translocated to the equatorial segment of acrosomal region during the acrosome reaction. On the other hand, severe damages were observed in the acrosomes of 60% of the cryopreserved spermatozoa. Localization of IZUMO1 in these spermatozoa was pattern P2 (IZUMO1 in whole equatorial segment), P3 (IZUMO1 in whole acrosomal region), or P4 (IZUMO was lost). Moreover, after the incubation to compare the stability of acrosomes and IZUMO1 localization between cryopreserved spermatozoa and freshly ejaculated spermatozoa, much more spermatozoa lost acrosomes and IZUMO1 in the cryopreserved samples compared with freshly ejaculated samples. These findings indicate that impaired

  8. Binding of Sperm to the Zona Pellucida Mediated by Sperm Carbohydrate-Binding Proteins is not Species-Specific in vitro between Pigs and Cattle

    PubMed Central

    Takahashi, Kazuya; Kikuchi, Kazuhiro; Uchida, Yasuomi; Kanai-Kitayama, Saeko; Suzuki, Reiichiro; Sato, Reiko; Toma, Kazunori; Geshi, Masaya; Akagi, Satoshi; Nakano, Minoru; Yonezawa, Naoto

    2013-01-01

    Carbohydrates are candidates for the basis of species-selective interaction of gametes during mammalian fertilization. In this study, we sought to clarify the roles of sugar residues in the species-selective, sperm–oocyte interaction in pigs and cattle. Acrosome-intact porcine and bovine sperm exhibited their strongest binding affinities for β-Gal and α-Man residues, respectively. Porcine-sperm specificity changed from β-Gal to α-Man after the acrosome reaction, while bovine-sperm specificity did not. Binding of acrosome-intact and acrosome-reacted sperm decreased after trypsinization, indicating that the carbohydrate-binding components are proteins. While immature oocytes bound homologous sperm preferentially to heterologous sperm, oocytes matured in vitro bound similar numbers of homologous and heterologous sperm. Lectin staining revealed the aggregation of α-Man residues on the outer surface of the porcine zona during maturation. In both species, zona-free, mature oocytes bound homologous sperm preferentially to heterologous sperm. The lectin-staining patterns of the zona pellucida and zona-free oocytes coincided with the carbohydrate-binding specificities of acrosome-intact and acrosome-reacted sperm, respectively, supporting the involvement of carbohydrates in gamete recognition in pigs and cattle. These results also indicate that sperm-zona pellucida and sperm–oolemma bindings are not strictly species-specific in pigs and cattle, and further suggest that sperm penetration into the zona and/or fusion with oolemma may be species-specific between pigs and cattle. PMID:24970158

  9. Ultrastructural and DNA fragmentation analyses in swim-up selected human sperm.

    PubMed

    Piomboni, P; Bruni, E; Capitani, S; Gambera, L; Moretti, E; La Marca, A; De Leo, V; Baccetti, B

    2006-01-01

    Seventeen sperm samples were evaluated by transmission electron microscopy (TEM) before and after swim-up separation. DNA-fragmentation was tested by terminal d-UTP nick end labeling (TUNEL) in unselected and selected semen samples, and the results were analyzed in relation to sperm ultrastructural characteristics detected by TEM. A significant improvement in mean numbers and percentages of structurally normal sperm was observed after swim-up selection, corresponding to a significant decrease in the percentage of necrotic and apoptotic sperm, while the percentage of sperm with immature nuclei did not change significantly. TUNEL indicated a significant decrease in chromatin-fragmented sperm after swim-up. Swim up selection based on sperm motility excludes many sperm with ultrastructural evidence of necrosis (absent or reacted acrosome, disrupted chromatin, broken plasma membrane) and apoptosis (misshapen nuclei with marginated chromatin), as confirmed by TUNEL analysis. Nevertheless, immature sperm with elliptical or roundish nuclei, misshapen acrosomes and uncondensed chromatin remain part of fertilizing pool.

  10. Proteasome activity and its relationship with protein phosphorylation during capacitation and acrosome reaction in human spermatozoa.

    PubMed

    Morales, Patricio; Díaz, Emilce S; Kong, Milene

    2007-01-01

    We have shown that the proteasome is present in mammalian sperm and plays a role during fertilisation. In this work we studied the relationship between protein phosphorylation and proteasomal activity in human sperm. Aliquots of motile sperm were incubated for 0, 5 and 18 h at 37 degrees C, 5% CO2, with different concentration of the kinase inhibitors genistein, H89 or tamoxifen. Control aliquots were treated with the inhibitor solvent. The chymotrypsin-like activity of the proteasome was assayed using a fluorogenic substrate. Aliquots of spermatozoa capacitated during 18 h were incubated for 30 min with kinase inhibitors and then with 7 microM progesterone (P). The percentage of viable acrosome-reacted sperm was evaluated using FITC-labeled Pisum sativum agglutinin. The results indicate that spermatozoa treated with different concentrations of genistein and tamoxifen did not modify the chymotrypsin-like activity of the proteasome during capacitation. On the other hand, proteasome activity was significantly decreased by incubation with H89. Sperm treatment with genistein, H89 and tamoxifen significantly inhibited the P-induced acrosome reaction. Western blot analysis indicated that the proteasome inhibitor, epoxomicin, reduced serine protein phosphorylation. These results suggest that the enzymatic activity of the proteasome is modulated by protein kinase A, and that both enzymes are involved in the P-induced acrosome reaction.

  11. Equatorin is not essential for acrosome biogenesis but is required for the acrosome reaction

    SciTech Connect

    Hao, Jianxiu; Chen, Min; Ji, Shaoyang; Wang, Xiaona; Wang, Yanbo; Huang, Xingxu; Yang, Lin; Wang, Yaqing; Cui, Xiuhong; Lv, Limin; Liu, Yixun; Gao, Fei

    2014-02-21

    Highlights: • Eqtn knockout mice were used for these experiments. • In vivo and in vitro fertilization analyses were performed. • Eqtn-deficient sperm were evaluated by transmission electron microscopy (TEM) and an A23187-induced acrosome reaction (AR) assay. • Co-immunoprecipitation (Co-IP) was performed to assess the interaction between Eqtn and the SNARE complex. - Abstract: The acrosome is a specialized organelle that covers the anterior part of the sperm nucleus and plays an essential role in mammalian fertilization. However, the regulatory mechanisms controlling acrosome biogenesis and acrosome exocytosis during fertilization are largely unknown. Equatorin (Eqtn) is a membrane protein that is specifically localized to the acrosomal membrane. In the present study, the physiological functions of Eqtn were investigated using a gene knockout mouse model. We found that Eqtn{sup −/−} males were subfertile. Only approximately 50% of plugged females were pregnant after mating with Eqtn{sup −/−} males, whereas more than 90% of plugged females were pregnant after mating with control males. Sperm and acrosomes from Eqtn{sup −/−} mice presented normal motility and morphology. However, the fertilization and induced acrosome exocytosis rates of Eqtn-deficient sperm were dramatically reduced. Further studies revealed that the Eqtn protein might interact with Syntaxin1a and SNAP25, but loss of Eqtn did not affect the protein levels of these genes. Therefore, our study demonstrates that Eqtn is not essential for acrosome biogenesis but is required for the acrosome reaction. Eqtn is involved in the fusion of the outer acrosomal membrane and the sperm plasma membrane during the acrosome reaction, most likely via an interaction with the SNARE complex.

  12. Localization and characterization of the acrosomal antigen recognized by GB24 on human spermatozoa.

    PubMed

    Fénichel, P; Dohr, G; Grivaux, C; Cervoni, F; Donzeau, M; Hsi, B L

    1990-10-01

    GB24, a mouse monoclonal antibody, recognizes a trophoblast-leukocyte cross-reactive antigen (TLX), which is likely identical to the membrane cofactor protein (MCP), a complement regulatory protein. GB24 reacts also with a human acrosomal sperm antigen (Fénichel et al.: J Reprod Fertil 87:699-706, 1989). By immunofluorescence or immunoperoxidase, testicular, epididymal, and ejaculated spermatozoa were found to be positive after fixation by acetone. Motile, suspended spermatozoa became positive only through conditions known to induce acrosome reaction (A23187, follicular fluid, contact with oocytes). Ultrastructural studies with immunogold staining localized this protein on the inner acrosome membrane and in the acrosomal content. By SDS-polyacrylamide gel electrophoresis, GB24 immunoprecipitated a unique protein of 48 kDa from capacitated and A23187-induced spermatozoa under reducing conditions. No cross-reactivity was found with mouse, boar, or ram spermatozoa. Localization of this human sperm antigen recognized by GB24 and its similarity with the TLX-MCP family antigens would suggest a possible role of this molecule during fertilization in sperm-egg binding or immune protection.

  13. A mechanism for differential release of acrosomal enzymes during the acrosome reaction.

    PubMed Central

    Hardy, D M; Oda, M N; Friend, D S; Huang, T T

    1991-01-01

    To study the organization of fertilization enzymes in the sperm acrosome, we isolated and characterized two physicochemically distinct acrosomal fractions of guinea-pig spermatozoa. A soluble fraction contained the 25,000-Mr acrosomal autoantigen, AA1, and most of the acrosomal hyaluronidase and dipeptidyl peptidase II activity. A particulate fraction, designated acrosomal matrix (AM), consisted of membraneless crescent-shaped structures, and contained most of the acrosomal proacrosin. The AM also contained a 28,000-Mr putative proacrosin-binding protein, and a very-high-Mr component which, on reduction, was dissociated into 48,000-Mr and 67,000-Mr subunits. Autoproteolytic dissolution of the AM correlated with proteolysis by acrosin of the 28,000-Mr and 48,000-Mr AM molecules. Components of both the AM and the soluble fraction were localized by immuno-electron microscopy to the electron-dense region of the guinea-pig sperm acrosome. We conclude that acrosomal molecules are segregated into soluble and matrix compartments. This segregation is a function of disulphide bonding and non-covalent interactions among the relatively few components of the AM. Association of acrosin with the AM may be the mechanism by which this enzyme's release from the spermatozoon during the acrosome reaction is delayed relative to the release of other acrosomal molecules. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:1903927

  14. Characterization of the active site of monkey sperm hyaluronidase.

    PubMed

    Yudin, A I; Li, M W; Robertson, K R; Cherr, G N; Overstreet, J W

    2001-05-01

    The mammalian sperm hyaluronidase, PH-20, is active in macaque spermatozoa at neutral and acid pH. Antibodies were produced to synthesized peptides representing regions of PH-20 that may be involved in hyaluronidase activity and designated peptide 1 (amino acid sequence 142-172) and peptide 3 (amino acid sequence 277-297). Western blotting of proteins extracted from the surface of acrosome-intact spermatozoa showed that the two peptide-specific, affinity-purified IgGs label a 64 kDa band corresponding to the PH-20 molecule. Western blots of acrosome-reacted spermatozoa showed that, under reducing conditions, the two anti-peptide IgGs label the 44 kDa band only, which represents the N-terminal fragment of PH-20. Anti-peptide 3 IgG also labels the 53 kDa form of PH-20 in extracts of acrosome-reacted spermatozoa. Peptide-specific, affinity-purified Fab fragments from both IgGs were shown by fluorescence microscopy and transmission electron microscopy to label the sperm plasma membrane, fused acrosomal vesicles, acrosomal matrix and inner acrosomal membrane. Fab fragments of anti-peptide 1 IgG, but not anti-peptide 3 IgG, inhibited hyaluronidase activity of PH-20 from the sperm surface and from extracts of acrosome-reacted spermatozoa at pH 7.0. Fab fragments of both anti-peptide IgGs inhibited sperm hyaluronidase activity at pH 5.0. It is concluded that the region of PH-20 encompassed by the amino acid sequence 142-172 is essential for hyaluronidase activity at neutral pH, whereas the region of amino acid sequence 277-297 may be more important at a lower pH. It is likely that these two regions are the acid/base catalyst site and the nucleophilic site, respectively, of PH-20 hyaluronidases.

  15. Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa

    PubMed Central

    Hossepian de Lima, Vera F. M.; Levenhagen, Marcelo A.; dos Santos, Ricarda M.; Assumpção, Terezinha I.; Jacomini, José O.; de Andrade, André F. C.; de Arruda, Rubens P.; Beletti, Marcelo E.

    2011-01-01

    The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized. PMID:21897100

  16. Transmission electron microscopy for characterization of acrosomal damage after Percoll gradient centrifugation of cryopreserved bovine spermatozoa.

    PubMed

    Oliveira, Letícia Z; Hossepian de Lima, Vera Fm; Levenhagen, Marcelo A; Santos, Ricarda M; Assumpção, Terezinha I; Jacomini, José O; Andrade, André Fc; Arruda, Rubens P; Beletti, Marcelo E

    2011-09-01

    The objective of this study was to characterize acrosomal ultrastructure following discontinuous Percoll gradient centrifugation of cryopreserved bovine sperm. Semen was collected from six bulls of different breeds and three ejaculates per bull were evaluated. Frozen semen samples were thawed and the acrosomal region of sperm cells was evaluated by transmission electron microscopy (TEM) before (n = 18) and after (n = 18) Percoll centrifugation. The evaluation of 20 sperm heads from each of the 36 samples analyzed ensured that a large number of cells were investigated. The data were subjected to analysis of variance at a level of significance of 5%. Percoll centrifugation reduced the percentage of sperm exhibiting normal acrosomes (from 61.77 to 30.24%), reduced the percentage of sperm presenting atypical acrosome reactions (from 28.38 to 4.84%) and increased the percentage of sperm exhibiting damage in the acrosome (from 6.14 to 64.26%). The percentage of sperm with typical acrosome reactions was not significantly different before (3.70%) and after (0.67%) centrifugation. TEM distinguished four different types of acrosomal status and enabled ultrastructural characterization of acrosomal injuries. The percentage of sperm exhibiting normal acrosomes decreased and damage in the acrosome was the most frequent acrosomal injury with the Percoll gradient centrifugation protocol utilized.

  17. Combined Effect of Trolox and EDTA on Frozen-Thawed Sperm Quality

    PubMed Central

    Keshtgar, Sara; Iravanpour, Farideh; Gharesi-Fard, Behrooz; Kazerooni, Marjaneh

    2016-01-01

    The freezing and thawing process not only is associated with serious damage to sperm such as damage to the plasma membrane and the acrosomal membrane but also changes the membrane permeability to some ions including calcium. Also, the generation of oxygen free radicals is increased during the freezing-thawing process. The purpose of this study was to evaluate of the effects of Trolox as an antioxidant and edetic acid (EDTA) as a calcium chelator on frozen-thawed (FT) sperm and compare these effects with those on fresh sperm. This study was done on these men of 25 healthy men, who referred to Shiraz Infertility Centerbetween2012 and2013. Normal samples were transferred to the ReproductivePhysiology Laboratory, Department of Physiology, Shiraz University of Medical Sciences, Shiraz. The samples were divided into two groups randomly: fresh and FT sperm groups. Each group was divided into five subgroups: control group, the solvent group (0.1%dimethyl sulfoxide [DMSO]), Trolox group (200μM), EDTA group (1.1mM), and Trolox+EDTA group. The percentages of motility, viability, and acrosome-reacted sperm were tested. The percentages of motility and viability in the FT sperm were lower than those in the fresh sperm. The progressive motility of the FT sperm was improved nonsignificantly with Trolox+EDTA. However, the effect of Trolox+EDTA on the progressive motility of the FT sperm was much more than that on the fresh sperm. The fewest acrosome-reacted sperm were observed in the EDTA-containingFT sperm. Antioxidant supplementation or omission of extracellular calcium may partly improve motility and also reduce acrosomal damage in FT sperm. PMID:27217608

  18. Hypercholesterolemia Impaired Sperm Functionality in Rabbits

    PubMed Central

    Monclus, Maria A.; Cabrillana, Maria E.; Clementi, Marisa A.; Espínola, Leandro S.; Cid Barría, Jose L.; Vincenti, Amanda E.; Santi, Analia G.; Fornés, Miguel W.

    2010-01-01

    Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a “folded head”-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events. PMID:20976152

  19. Correlation between tyrosine phosphorylation intensity of a 107 kDa protein band and A23187-induced acrosome reaction in human spermatozoa.

    PubMed

    Picherit-Marchenay, C; Bréchard, S; Boucher, D; Grizard, G

    2004-12-01

    This study, performed using semen samples from 10 men, investigated the relationship between sperm protein tyrosine phosphorylation and acrosomal status in conditions supporting in vitro capacitation. Percoll-selected spermatozoa (cells from the 95% fraction) were incubated for 3 h at 37 degrees C under an atmosphere of 5% CO2 in air, in a polyvinyl alcohol (1 mg ml(-1)) containing Biggers-Whitten-Whittingham's medium, nonsupplemented or supplemented with either bovine serum albumin (BSA; fatty acid free, 3 mg ml(-1)) or 2-hydroxy-propyl-beta-cyclodextrin (2-OH-p-beta-CD; 0.5, 1, 2 mmol l(-1)). Sperm suspension in each medium was split into two aliquots. The first was used to evaluate the acrosomal status by staining with the fluorescein isothiocyanate Pisum sativum agglutinin after induction of the acrosome reaction (AR) for 45 min with 10 micromol l(-1) of A23187 calcium ionophore. The second aliquot was used for sodium dodecyl sulphate polyacrylamide gel electrophoresis and immunoblotting, followed by a densitometric analysis. Compared with the nonsupplemented medium, BSA- or 2-OH-p-beta-CD-supplementation induced an increase in both the percentage of live acrosome-reacted sperm and the tyrosine phosphorylation intensity of the main phosphorylated 107 kDa protein. A correlation between the percentage of live acrosome-reacted sperm and the 107-kDa protein phosphotyrosine intensity was observed. Therefore, the 107 kDa protein-phosphotyrosine level measurement would bring additional information to conventional semen parameters in the assessment of the human sperm functionality.

  20. Induction of ultra-morphological features of apoptosis in mature and immature sperm.

    PubMed

    Grunewald, Sonja; Fitzl, Guenther; Springsguth, Christopher

    2017-01-01

    There is a fundamental body of evidence suggesting that activated apoptosis signaling in ejaculated human sperm negatively influences their fertilization potential. However, it is still controversial whether this apoptotic signaling is a relic of an abortive apoptosis related to spermatogenesis or if it should be regarded as a functional preformed pathway in mature sperm leading to stereotypical morphological changes reflecting nuclear disassembly. To address this question, apoptosis was induced using betulinic acid in mature and immature ejaculated human sperm enriched by density gradient centrifugation. Execution of apoptosis was monitored by observing ultra-morphological changes via transmission electron microscopy. Typical morphological signs of apoptosis in somatic cells include plasma membrane blebbing with the formation of apoptotic bodies, impaired mitochondrial integrity, defects of the nuclear envelope, and nuclear fragmentation; these morphologies have also been observed in human sperm. In addition, these apoptotic characteristics were more frequent in immature sperm compared to mature sperm. Following betulinic acid treatment, apoptosis-related morphological changes were induced in mature sperm from healthy donors. This effect was much less pronounced in immature sperm. Moreover, in both fractions, the betulinic acid treatment increased the percentage of acrosome-reacted sperm. The results of our ultra-morphological study prove the functional competence of apoptosis in mature ejaculated human sperm. The theory of a sole abortive process may be valid only for immature sperm. The induction of the acrosome reaction by stimulating apoptosis might shed light on the biological relevance of sperm apoptosis.

  1. Fucosyl neoglycoprotein binds to mouse epididymal spermatozoa and inhibits sperm binding to the egg zona pellucida.

    PubMed

    Oh, Y S; Ahn, H S; Gye, M C

    2013-12-01

    Glycan epitopes of cellular glycoconjugates act as versatile biochemical signals, and this sugar coding plays an important role in cell-to-cell recognition processes. In this study, our aims were to determine the distribution of sperm receptors with activity for fucosyl- and galactosyl glycans and to address whether monosugar neoglycoproteins functionally mimic the binding between zona pellucida (ZP) glycoproteins and spermatozoa. In mouse epididymal spermatozoa with intact acrosomes, fucopyranosyl bovine serum albumin (BSA-Fuc) bound to the segment of the acrosome, the equatorial segment, and the postacrosome region of the sperm head. Galactosyl BSA (BSA-Gal) binding activity was similar to that of BSA-Fuc, but was weaker. In acrosome-reacted spermatozoa treated with the Ca(2+) ionophore A23187, BSA-zuc binding was lost in the apical segment of the acrosome but remained in the equatorial segment and postacrosome regions. BSA-Gal binding to the equatorial region was increased. In the presence of 2.5 μg ml(-1) BSA-Fuc, in vitro sperm-ZP binding was significantly decreased, indicating that fucosyl BSA functionally mimics ZP glycoproteins during sperm-egg ZP interactions. At the same concentration, BSA-Gal was not effective. Fucosyl BSA that efficiently inhibited the sperm-ZP binding can mimic the ZP glycoconjugate and has potential for use as a sperm fertility control agent in mouse.

  2. Dynamic regulation of sperm interactions with the zona pellucida prior to and after fertilisation.

    PubMed

    Gadella, B M

    2012-01-01

    Recent findings have refined our thinking on sperm interactions with the cumulus-oocyte complex (COC) and our understanding of how, at the molecular level, the sperm cell fertilises the oocyte. Proteomic analyses has identified a capacitation-dependent sperm surface reordering that leads to the formation of functional multiprotein complexes involved in zona-cumulus interactions in several mammalian species. During this process, multiple docking of the acrosomal membrane to the plasma membrane takes place. In contrast with the dogma that the acrosome reaction is initiated when spermatozoa bind to the zona pellucida (ZP), it has been established recently that, in mice, the fertilising spermatozoon initiates its acrosome reaction during its voyage through the cumulus before it reaches the ZP. In fact, even acrosome-reacted mouse spermatozoa collected from the perivitelline space can fertilise another ZP-intact oocyte. The oviduct appears to influence the extracellular matrix properties of the spermatozoa as well as the COC. This may influence sperm binding and penetration of the cumulus and ZP, and, in doing so, increase monospermic while decreasing polyspermic fertilisation rates. Structural analysis of the ZP has shed new light on how spermatozoa bind and penetrate this structure and how the cortical reaction blocks sperm-ZP interactions. The current understanding of sperm interactions with the cumulus and ZP layers surrounding the oocyte is reviewed with a special emphasis on the lack of comparative knowledge on this topic in humans, as well as in most farm mammals.

  3. Equatorial segment protein (ESP) is a human alloantigen involved in sperm-egg binding and fusion.

    PubMed

    Wolkowicz, M J; Digilio, L; Klotz, K; Shetty, J; Flickinger, C J; Herr, J C

    2008-01-01

    The equatorial segment of the sperm head is known to play a role in fertilization; however, the specific sperm molecules contributing to the integrity of the equatorial segment and in binding and fusion at the oolemma remain incomplete. Moreover, identification of molecular mediators of fertilization that are also immunogenic in humans is predicted to advance both the diagnosis and treatment of immune infertility. We previously reported the cloning of Equatorial Segment Protein (ESP), a protein localized to the equatorial segment of ejaculated human sperm. ESP is a biomarker for a subcompartment of the acrosomal matrix that can be traced through all stages of acrosome biogenesis (Wolkowicz et al, 2003). In the present study, ESP immunoreacted on Western blots with 4 (27%) of 15 antisperm antibody (ASA)-positive serum samples from infertile male patients and 2 (40%) of 5 ASA-positive female sera. Immunofluorescent studies revealed ESP in the equatorial segment of 89% of acrosome-reacted sperm. ESP persisted as a defined equatorial segment band on 100% of sperm tightly bound to the oolemma of hamster eggs. Antisera to recombinant human ESP inhibited both oolemmal binding and fusion of human sperm in the hamster egg penetration assay. The results indicate that ESP is a human alloantigen involved in sperm-egg binding and fusion. Defined recombinant sperm immunogens, such as ESP, may offer opportunities for differential diagnosis of immune infertility.

  4. A role for carbohydrate recognition in mammalian sperm-egg binding

    SciTech Connect

    Clark, Gary F.

    2014-08-01

    Highlights: • Mammalian sperm-egg binding as a carbohydrate dependent species recognition event. • The role of carbohydrate recognition in human, mouse and pig sperm-egg binding. • Historical perspective and future directions for research focused on gamete binding. - Abstract: Mammalian fertilization usually requires three sequential cell–cell interactions: (i) initial binding of sperm to the specialized extracellular matrix coating the egg known as the zona pellucida (ZP); (ii) binding of sperm to the ZP via the inner acrosomal membrane that is exposed following the induction of acrosomal exocytosis; and (iii) adhesion of acrosome-reacted sperm to the plasma membrane of the egg cell, enabling subsequent fusion of these gametes. The focus of this review is on the initial binding of intact sperm to the mammalian ZP. Evidence collected over the past fifty years has confirmed that this interaction relies primarily on the recognition of carbohydrate sequences presented on the ZP by lectin-like egg binding proteins located on the plasma membrane of sperm. There is also evidence that the same carbohydrate sequences that mediate binding also function as ligands for lectins on lymphocytes that can inactivate immune responses, likely protecting the egg and the developing embryo up to the stage of blastocyst hatching. The literature related to initial sperm-ZP binding in the three major mammalian models (human, mouse and pig) is discussed. Historical perspectives and future directions for research related to this aspect of gamete adhesion are also presented.

  5. Autoantigen 1 of the guinea pig sperm acrosome is the homologue of mouse Tpx-1 and human TPX1 and is a member of the cysteine-rich secretory protein (CRISP) family.

    PubMed

    Foster, J A; Gerton, G L

    1996-06-01

    We have cloned and sequenced cDNAs encoding autoantigen 1 (AA1), a testis-specific protein and the major autoantigen of the guinea pig sperm acrosome. The cDNA predicts a precursor protein of 244 amino acids including a 21 amino acid hydrophobic, secretory signal sequence. The mature polypeptide is predicted to have a molecular mass of 24,891 Daltons which agrees with the experimentally determined molecular weight of 25,000. Consistent with previous studies demonstrating that AA1 is not a glycoprotein, the predicted amino acid sequence contained no canonical sites for N-linked glycosylation. Comparison with other sequences showed that AA1 is the guinea pig homologue of the testis-specific protein Tpx-1 in mice and TPX1 in humans. AA1 also showed significant amino acid sequence homology with other cysteine-rich secretory proteins (CRISP's): rat and mouse acidic epididymal glycoproteins (AEG; also known as proteins D/E in rats) and helothermine, a toxin from the Mexican beaded lizard. In addition, AA1 had a lesser degree of homology with antigen 5 (vespid wasp venom), PR-1 (a plant pathogenesis related protein), and GliPR (a protein identified in human gliomas). Northern analysis of RNA from purified guinea pig spermatogenic cells showed that a 1.5 kb message was first detected in pachytene spermatocytes, was strongest in round spermatids, and was detected at a low level in condensing spermatids. Immunoblot analysis and metabolic labeling data of AA1 in spermatogenic cells showed that the protein was synthesized as early as the pachytene spermatocyte stage of spermatogenesis. Thus, the patterns of AA1 mRNA and protein expression during spermatogenesis are similar to the expression of other acrosomal mRNAs and proteins that are first detected meiotically.

  6. Changes in exposed membrane proteins during in vitro capacitation of boar sperm

    SciTech Connect

    Berger, T. )

    1990-11-01

    Exposed plasma membrane proteins were labeled with {sup 125}I before and after incubation of boar sperm under capacitating conditions. Labeled protein profiles were compared to the ability of the sperm to penetrate zona-free hamster ova. Quantitatively, the labeled sperm membrane proteins were primarily low Mr prior to capacitation. The majority of the labeled seminal plasma protein was also low Mr. After capacitation, two new proteins (64,000 Mr and 78,000 Mr) were labeled. Sperm did not exhibit these exposed membrane proteins when incubated under noncapacitating conditions. Appearance of these proteins was not correlated to the percentage of acrosome-reacted sperm. Although the 64,000 Mr protein was not consistently observed, the relative labeling of the 78,000 Mr protein was highly correlated with the ability of sperm to fuse with zona-free hamster ova. The 78,000 Mr protein may be a sperm protein involved in fusion with the egg plasma membrane.

  7. Influence of temperature and sperm preparation on the quality of spermatozoa.

    PubMed

    Thijssen, Annelies; Klerkx, Elke; Huyser, Carin; Bosmans, Eugene; Campo, Rudi; Ombelet, Willem

    2014-04-01

    This study investigated the effects of long-term (24h) in-vitro sperm incubation at room temperature (RT; 23°C) versus testis temperature (35°C) on various sperm-quality parameters. Semen samples (n=41) were prepared both by density-gradient centrifugation (DGC) and the swim-up technique in order to compare the influence of sperm preparation on sperm quality after incubation. Progressive motility and morphology were significantly higher after incubation at RT compared with 35°C (P<0.001 and P<0.01, respectively). The proportions of acrosome-reacted, apoptotic and dead spermatozoa were significantly lower in samples incubated for 24h at RT compared with 35°C (P<0.001, P=0.01 and P<0.001, respectively). The number of motile, morphologically normal, non-acrosome-reacted and nonapoptotic spermatozoa recovered after sperm preparation was significantly higher in DGC compared with swim-up samples (P<0.001). However, spermatozoa prepared by swim-up showed better survival after incubation compared with DGC-prepared spermatozoa, especially when incubated at 35°C. In conclusion, this study indicates a significantly better and longer preservation of sperm quality when incubation is performed at RT. These findings may convince laboratories to change the routinely used sperm storage conditions in order to maximize the quality of the prepared sperm sample.

  8. Effect of various commercial buffers on sperm viability and capacitation.

    PubMed

    Andrisani, Alessandra; Donà, Gabriella; Ambrosini, Guido; Bonanni, Guglielmo; Bragadin, Marcantonio; Cosmi, Erich; Clari, Giulio; Armanini, Decio; Bordin, Luciana

    2014-08-01

    A wide variety of sperm preparation protocols are currently available for assisted conception. They include density gradient separation and washing methods. Both aim at isolating and capacitating as much motile sperm as possible for subsequent oocyte fertilization. The aim of this study was to examine the effects of four commercial sperm washing buffers on sperm viability and capacitation. Semen samples from 48 healthy donors (normal values of sperm count, motility, morphology, and volume) were analyzed. After separation (density gradient 40/80%), sperm were incubated in various buffers then analysed for reactive oxygen species (ROS) production, viability, tyrosine phosphorylation (Tyr-P), cholera toxin B subunit (CTB) labeling, and the acrosome reaction (AR). The buffers affected ROS generation in various ways resulting either in rapid cell degeneration (when the amount of ROS was too high for cell survival) or the inability of the cells to maintain correct functioning (when ROS were too few). Only when the correct ROS generation curve was maintained, suitable membrane reorganization, evidenced by CTB labeling was achieved, leading to the highest percentages of both Tyr-P- and acrosome-reacted-cells. Distinguishing each particular pathological state of the sperm sample would be helpful to select the preferred buffer treatment since both ROS production and membrane reorganization can be significantly altered by commercial buffers.

  9. Sperm quality improvement after date seed oil in vitro supplementation in spontaneous and induced oxidative stress

    PubMed Central

    Fatma, Ben A.; Nozha, Chakroun F.; Ines, Dammak; Hamadi, Attia; Basma, Hentati; Leila, Ammar K.

    2009-01-01

    In vitro supplementation with date seed oil (DSO) can protect spermatozoa against hydrogen peroxide (H2O2)-mediated damage and can improve sperm function, possibly owing to antioxidant properties. We tested the antioxidant effects of DSO on human sperm motility, sperm viability, reacted acrosome and lipid peroxidation assessed in vitro after H2O2-mediated oxidative damage in spermatozoa. Sixteen patients (mean age: 35 years; range: 25–45 years) referred to the Histology–Embryology Laboratory of the Medicine Faculty of Sfax for semen analysis after 12–24 months of sexual intercourse without conception were selected. After spermiogram, sperm selection by two-interface discontinuous Sill Select gradient was performed, and selected spermatozoa were used in four experimental assays: control; incubation with 100 μm H2O2; incubation with 0.1% DSO; and co-incubation with 0.1% DSO and 100 μm H2O2. Motility and viability were determined using World Health Organization criteria. Acrosome reaction and lipid peroxidation were assessed by staining with fluorescein isothiocyanate-Pisum sativum and spectrophotometric measurement of malondialdehyde, respectively. Results showed that incubation with H2O2 alone led to a significant increase in lipid peroxidation (57.83%, P < 0.05) associated with a significant decrease in sperm motility, sperm viability (after 30 min and 24 h) and percentage of reacted acrosome (P < 0.05). Date seed oil improved sperm motility after 24 h of incubation (P < 0.05) and protected spermatozoa against the deleterious effects of H2O2 on motility, viability, acrosome reaction and lipid peroxidation. We conclude that supplementation with DSO may have a function in antioxidant protection against male infertility. PMID:19151733

  10. MARCKS Protein Is Phosphorylated and Regulates Calcium Mobilization during Human Acrosomal Exocytosis

    PubMed Central

    Rodriguez Peña, Marcelo J.; Castillo Bennett, Jimena V.; Soler, Osvaldo M.; Mayorga, Luis S.; Michaut, Marcela A.

    2013-01-01

    Acrosomal exocytosis is a calcium-regulated exocytosis that can be triggered by PKC activators. The involvement of PKC in acrosomal exocytosis has not been fully elucidated, and it is unknown if MARCKS, the major substrate for PKC, participates in this exocytosis. Here, we report that MARCKS is expressed in human spermatozoa and localizes to the sperm head and the tail. Calcium- and phorbol ester-triggered acrosomal exocytosis in permeabilized sperm was abrogated by different anti-MARCKS antibodies raised against two different domains, indicating that the protein participates in acrosomal exocytosis. Interestingly, an anti-phosphorylated MARCKS antibody was not able to inhibit secretion. Similar results were obtained using recombinant proteins and phospho-mutants of MARCKS effector domain (ED), indicating that phosphorylation regulates MARCKS function in acrosomal exocytosis. It is known that unphosphorylated MARCKS sequesters PIP2. This phospholipid is the precursor for IP3, which in turn triggers release of calcium from the acrosome during acrosomal exocytosis. We found that PIP2 and adenophostin, a potent IP3-receptor agonist, rescued MARCKS inhibition in permeabilized sperm, suggesting that MARCKS inhibits acrosomal exocytosis by sequestering PIP2 and, indirectly, MARCKS regulates the intracellular calcium mobilization. In non-permeabilized sperm, a permeable peptide of MARCKS ED also inhibited acrosomal exocytosis when stimulated by a natural agonist such as progesterone, and pharmacological inducers such as calcium ionophore and phorbol ester. The preincubation of human sperm with the permeable MARCKS ED abolished the increase in calcium levels caused by progesterone, demonstrating that MARCKS regulates calcium mobilization. In addition, the phosphorylation of MARCKS increased during acrosomal exocytosis stimulated by the same activators. Altogether, these results show that MARCKS is a negative modulator of the acrosomal exocytosis, probably by sequestering

  11. Structure of the acrosomal bundle.

    PubMed

    Schmid, Michael F; Sherman, Michael B; Matsudaira, Paul; Chiu, Wah

    2004-09-02

    In the unactivated Limulus sperm, a 60- micro m-long bundle of actin filaments crosslinked by the protein scruin is bent and twisted into a coil around the base of the nucleus. At fertilization, the bundle uncoils and fully extends in five seconds to support a finger of membrane known as the acrosomal process. This biological spring is powered by stored elastic energy and does not require the action of motor proteins or actin polymerization. In a 9.5-A electron cryomicroscopic structure of the extended bundle, we show that twist, tilt and rotation of actin-scruin subunits deviate widely from a 'standard' F-actin filament. This variability in structural organization allows filaments to pack into a highly ordered and rigid bundle in the extended state and suggests a mechanism for storing and releasing energy between coiled and extended states without disassembly.

  12. Distribution, crypticity, stability, and localization of α-L-fucosidase of mouse cauda epididymal sperm.

    PubMed

    Phopin, Kamonrat; Nimlamool, Wutigri; Bartlett, Mackenzie J; Bean, Barry S

    2012-03-01

    Sperm-associated and semen-specific isoforms of α-L-fucosidase are thought to function in fertilization in numerous organisms. Here, we report the localization, distribution, crypticity, and stability of this enzyme in mouse cauda epididymal sperm and cauda fluid. Western analysis revealed that the sperm-associated α-L-fucosidase is present as two isoforms (Mr ∼49 and 56 kDa), whereas the cauda fluid α-L-fucosidase shows a single band at 50 kDa. α-L-Fucosidase activity was detected using the fluorogenic substrate 4-MU-FUC. Of the total α-L-fucosidase activity recovered in the cauda epididymal contents, 74% was found in the cell-free cauda fluid and about 7% was found in sperm cells. During capacitation or permeabilization, cryptic intracellular stores of soluble enzyme were released to the supernatant, while leaving bound enzyme concentrated within the small volume of sperm. Moreover, membrane-associated enzyme activity was still detectable in acrosome-reacted cells. Immunofluorescence studies support the presence of α-L-fucosidase (originally localizing at the acrosomal area) at the equatorial segment after the acrosome reaction. α-L-Fucosidase activity of both cauda fluid and sperm at 37°C, 5% CO(2) was relatively stable and detectable up to 72 hr. The stability and appearance of mouse sperm-associated α-L-fucosidase in the equatorial segment after the acrosome reaction suggest that α-L-fucosidase may be involved in sperm-egg interaction.

  13. Oviductosome-Sperm Membrane Interaction in Cargo Delivery

    PubMed Central

    Al-Dossary, Amal A.; Bathala, Pradeepthi; Caplan, Jeffrey L.; Martin-DeLeon, Patricia A.

    2015-01-01

    Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca2+ efflux pump, plasma membrane Ca2+ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4–64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes. PMID:26023236

  14. Ability of abnormally-shaped human spermatozoa to adhere to and penetrate zona-free hamster eggs: correlation with sperm morphology and postincubation motility.

    PubMed

    Bronson, Richard A; Bronson, Susan K; Oula, Lucila D

    2007-01-01

    A body of evidence indicates that morphologically abnormal human spermatozoa may exhibit impaired ability to fertilize. Yet teratospermia has widely varying etiologies, including associations with varicoceles, following fever, cigarette smoking, and exposure to polychlorinated biphenyls. Abnormalities of sperm shape in mice have also been shown to be associated with autosomal gene mutations. These varying causes of teratospermia could have different molecular consequences reflected in altered sperm function. We studied the ability of morphologically abnormal human sperm to penetrate zona-free hamster eggs as a measure of their ability to undergo an acrosome reaction and gamete membrane fusion. Motile sperm from ejaculates containing 15% normal sperm or less, as judged by World Health Organization (1999) criteria, were recovered by ISolate density centrifugation and capacitated by overnight incubation. Zona-free hamster eggs were inseminated with 1 x 10(6) motile capacitated cells and scored for sperm penetration after 3 hours of coincubation. A significant trend was found between the percent of abnormal spermatozoa within the ejaculate and impaired egg-penetrating ability, reflected in the percent of eggs penetrated, the number of penetrating sperm per egg, and the number of sperm adherent to the oolemma. Because only acrosome-reacted human spermatozoa adhere to the oolemma, these results support the notion that abnormally shaped sperm may exhibit an impaired ability to undergo an acrosome reaction. A correlation was also noted between the loss of motility of sperm following overnight incubation and impairment of their ability to undergo gamete membrane fusion. These results confirm prior findings at the level of the zona pellucida that abnormally shaped sperm exhibit functional abnormalities. However, a wide variation was observed between men in the behavior of such sperm, including occasionally high rates of egg penetration. These observations suggest that

  15. Sperm Competition, Sperm Numbers and Sperm Quality in Muroid Rodents

    PubMed Central

    Gómez Montoto, Laura; Magaña, Concepción; Tourmente, Maximiliano; Martín-Coello, Juan; Crespo, Cristina; Luque-Larena, Juan José

    2011-01-01

    Sperm competition favors increases in relative testes mass and production efficiency, and changes in sperm phenotype that result in faster swimming speeds. However, little is known about its effects on traits that contribute to determine the quality of a whole ejaculate (i.e., proportion of motile, viable, morphologically normal and acrosome intact sperm) and that are key determinants of fertilization success. Two competing hypotheses lead to alternative predictions: (a) sperm quantity and quality traits co-evolve under sperm competition because they play complementary roles in determining ejaculate's competitive ability, or (b) energetic constraints force trade-offs between traits depending on their relevance in providing a competitive advantage. We examined relationships between sperm competition levels, sperm quantity, and traits that determine ejaculate quality, in a comparative study of 18 rodent species using phylogenetically controlled analyses. Total sperm numbers were positively correlated to proportions of normal sperm, acrosome integrity and motile sperm; the latter three were also significantly related among themselves, suggesting no trade-offs between traits. In addition, testes mass corrected for body mass (i.e., relative testes mass), showed a strong association with sperm numbers, and positive significant associations with all sperm traits that determine ejaculate quality with the exception of live sperm. An “overall sperm quality” parameter obtained by principal component analysis (which explained 85% of the variance) was more strongly associated with relative testes mass than any individual quality trait. Overall sperm quality was as strongly associated with relative testes mass as sperm numbers. Thus, sperm quality traits improve under sperm competition in an integrated manner suggesting that a combination of all traits is what makes ejaculates more competitive. In evolutionary terms this implies that a complex network of genetic and

  16. ROLE OF THE GAMETE MEMBRANES IN FERTILIZATION IN SACCOGLOSSUS KOWALEVSKII (ENTEROPNEUSTA). I. THE ACROSOMAL REGION AND ITS CHANGES IN EARLY STAGES OF FERTILIZATION.

    PubMed

    COLWIN, A L; COLWIN, L H

    1963-12-01

    Previous electron microscope studies of sperm-egg association in the annelid Hydroides revealed novel aspects with respect to the acrosomal region. To determine whether these aspects were unique, a comparable study was made of a species belonging to a widely separated phylum, Hemichordata. Osmium tetroxide-fixed polyspermic material of the enteropneust, Saccoglossus, was used. The acrosomal region includes the membrane-bounded acrosome, with its large acrosomal granule and shallow adnuclear invagination, and the periacrosomal material which surrounds the acrosome except at the apex; here, the acrosomal membrane lies very close to the enclosing sperm plasma membrane. After reaching the egg envelope, the spermatozoon is activated and undergoes a series of changes: the apex dehisces and around the resulting orifice the acrosomal and sperm plasma membranes form a continuous mosaic membrane. The acrosomal granule disappears. Within 7 seconds the invagination becomes the acrosomal tubule, spans the egg envelopes, and meets the egg plasma membrane. The rest of the acrosomal vesicle everts. The periacrosomal mass changes profoundly: part becomes a fibrous core (possibly equivalent to a perforatorium); part remains as a peripheral ring. The basic pattern of structure and sperm-egg association in Saccoglossus is the same as in Hydroides. Previous evidence from four other phyla as interpreted here also indicates conformity to this pattern. The major role of the acrosome is apparently to deliver the sperm plasma membrane to the egg plasma membrane.

  17. Expression of α-gustducin and α-transducin, G proteins coupled with taste receptors, in boar sperm.

    PubMed

    Spinaci, M; Bucci, D; Mazzoni, M; Giaretta, E; Bernardini, C; Vallorani, C; Tamanini, C; Clavenzani, P; Galeati, G

    2014-07-01

    During the transit in the female genital tract, spermatozoa are exposed to an environment that varies in composition from the vagina to the oviduct. Because G proteins, α-gustducin and α-transducin, are accepted as markers of chemosensitive cells, this study was aimed at assessing whether these proteins are expressed in boar germ cells. Ejaculated sperm extracts were analyzed by Western blot, and indirect immunofluorescence was performed on testis sections, smears of epididymal and ejaculated spermatozoa, sperm cells after in vitro induction of capacitation and acrosome reaction (IVAR), and in sperm cells bound to zona pellucida during IVF. Based on immunoblot results, both G proteins are present in boar sperm. In the testicular tissue sections, α-gustducin and α-transducin positivity was recorded in the germinal cells near the tubular lumen, whereas no positive signal was evident in spermatogonia located in the outer region of the seminiferous tubules. α-Gustducin expression in epididymal and ejaculated spermatozoa was mainly detectable in both the acrosome and the principal piece of the tail, whereas α-transducin was confined to the acrosome and the midpiece. No changes after in vitro induction of capacitation and IVAR were observed, except for the disappearance of acrosomal positivity in reacted spermatozoa. In sperm bound to zona pellucida, the G protein signal was congruent with that observed in IVAR cells. To the best of our knowledge, this is the first description of α-transducin in mammalian sperm and the first description of α-gustducin in boar sperm. Further studies are needed to clarify the possible role of these G proteins in sperm physiology.

  18. Acrosome reaction in spermatozoa from the amphioxus Branchiostoma belcheri (Cephalochordata, Chordata).

    PubMed

    Morisawa, Sachiko; Mizuta, Takanobu; Kubokawa, Kaoru; Tanaka, Hiroyuki; Morisawa, Masaaki

    2004-11-01

    The formation of an acrosomal process at acrosomal exocytosis in spermatozoa of the amphioxus was described in the present report for the first time. A non-reacted acrosome was located in front of the nucleus, where a cup-shaped acrosomal vesicle covered a conical accumulation of subacrosomal material. When naturally spawned spermatozoa were treated with a calcium ionophore, ionomycin, the acrosomal vesicle opened at the apex and an acrosomal process was projected. The process exhibited a filamentous structure. The reaction followed the mode typically seen in marine invertebrates. These observations suggest that the features and function of the acrosome of amphioxus, whose position is on the border between invertebrates and vertebrates, reflect their ecological adaptation and phylogenic position.

  19. RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

    SciTech Connect

    Bello, Oscar D.; Zanetti, M. Natalia; Mayorga, Luis S.; Michaut, Marcela A.

    2012-03-10

    Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black

  20. Acrosome intactness and seminal hyaluronidase activity: relationship with conventional seminal parameters.

    PubMed

    Tambe, A S; Kaore, S B; Sawane, M V; Gosavi, G B

    2001-03-01

    Seminal hyaluronidase activity was estimated after liquefaction in semen samples of 100 male partners of infertile couples including 16 azoospermic (no spermatozoon) men and 48 fertility proven men by a method based on measurement of the area of digestion of substrate (hyaluronic acid) in agar plate. Semen samples were also evaluated for Acrosomal Intactness (AI) test except the azoospermics of the studied samples. Seminal hyaluronidase activity was completely absent in azoospermic specimens confirming its cellular origin. Seminal hyaluronidase activity was found to be significantly correlated, statistically, with sperm density (r = 0.708, p < 0.001), % motility (r = 0.6478, p < 0.001) and % normal sperm morphology (r = 0.5724, p < 0.001). Acrosomal Intactness (AI) test scores were also well correlated with sperm density (r = 0.6477, p < 0.001), % motility (r = 0.5965, p < 0.001) and % normal morphology (r = 0.6237, p < 0.001). Both values were higher in semen samples with normal routine parameters (proven fertility and normozoospermic infertile groups) than those compared with abnormal routine parameters (oligozoospermic). We also found very highly significant correlation (r = 0.8442) between seminal hyaluronidase activity and Acrosomal Intactness scores, statistically (p < 0.001). This could be because; normal germinal semineferous epithelium generates abundant number of sperms with normal motility and morphology that are also having intact acrosome. Intact acrosome prevents loss of acrosomal enzymatic activity (e.g. hyaluronidase) until released after liquefaction during seminal analysis and during acrosomal reaction in female genital tract prior to fertilization. Seminal hyaluronidase activity, thus determined, is primarily dependent upon the intact status of acrosome. As each sperm contributes to the seminal hyaluronidase activity, it is directly correlated with sperm density; but at the same time it exhibits goods correlation with % motility and % normal

  1. Mouse SLLP1, a sperm lysozyme-like protein involved in sperm-egg binding and fertilization.

    PubMed

    Herrero, María Belén; Mandal, Arabinda; Digilio, Laura C; Coonrod, Scott A; Maier, Bernhard; Herr, John C

    2005-08-01

    This study demonstrates the retention of mouse sperm lysozyme-like protein (mSLLP1) in the equatorial segment of spermatozoa following the acrosome reaction and a role for mSLLP1 in sperm-egg binding and fertilization. Treatment of cumulus intact oocytes with either recmSLLP1 or its antiserum resulted in a significant (P < or = 0.05) inhibition of fertilization. Co-incubation of zona-free mouse oocytes with capacitated mouse spermatozoa in the presence of varying concentrations of anti-recmSLLP1 serum or recmSLLP1 also inhibited sperm-oolemma binding. A complete inhibition of binding and fusion of spermatozoa to the oocyte occurred at 12.5 muM concentration of recmSLLP1, while conventional chicken and human lysozymes did not block sperm-egg binding. mSLLP1 showed receptor sites in the perivitelline space as well as on the microvillar region of the egg plasma membrane. The retention of mSLLP1 in the equatorial segment of acrosome-reacted sperm, the inhibitory effects of both recmSLLP1 and antibodies to SLLP1 on in vitro fertilization with both cumulus intact and zona-free eggs, and the definition of complementary SLLP1-binding sites on the egg plasma membrane together support the hypothesis that a c lysozyme-like protein is involved in the binding of spermatozoa to the egg plasma membrane during fertilization.

  2. Effect of divalent ions in acrosome reaction induced by glycosamineglycans in porcine spermatozoa.

    PubMed

    Delgado, N M; Carranco, A; Merchant, H; Reyes, R

    1985-01-01

    Magnesium, calcium, and zinc at the concentration of 10 microM are capable of inducing a "true" acrosome reaction in the pig spermatozoa judged by the criteria of the fusion of the acrosome and the plasmatic membrane at the anterior region or the sperm nucleus. The optimal percent of acrosome reaction reached by any of the ions tested as a whole was 50%. When glycosamineglycan sulfate (GAGs) plus 10 microM of Mg++, Ca++, or Zn++ was added, they reach to 70-80% of acrosome reaction. At the electrom microscope, thin sections taken from pig spermatozoa treated with ions, GAGs, or ion + GAGs under optimal experimental conditions revealed the same pattern of acrosomal reaction. Results suggest the important role that divalent cations play in general in the induction of the acrosome reaction and question the so-called essential role of calcium ions.

  3. Localized surface antigens of guinea pig sperm migrate to new regions prior to fertilization

    PubMed Central

    1984-01-01

    We have previously defined distinct localizations of antigens on the surface of the guinea pig sperm using monoclonal antibodies. In the present study we have demonstrated that these antigen localizations are dynamic and can be altered during changes in the functional state of the sperm. Before the sperm is capable of fertilizing the egg, it must undergo capacitation and an exocytic event, the acrosome reaction. Prior to capacitation, the antigen recognized by the monoclonal antibody, PT-1, was restricted to the posterior tail region (principle piece and end piece). After incubation in capacitating media at 37 degrees C for 1 h, 100% of the sperm population showed migration of the PT-1 antigen onto the anterior tail. This redistribution of surface antigen resulted from a migration of the surface molecules originally present on the posterior tail. It did not occur in the presence of metabolic poisons or when tail-beating was prevented. It was temperature-dependent, and did not require exogenous Ca2+. Since the PT- 1 antigen is freely diffusing on the posterior tail before migration, the mechanism of redistribution could involve the alteration of a presumptive membrane barrier. In addition, we observed the redistribution of a second surface antigen after the acrosome reaction. The antigen recognized by the monoclonal antibody, PH-20, was localized exclusively in the posterior head region of acrosome-intact sperm. Within 7-10 min of induction of the acrosome reaction with Ca2+ and A23187, 90-100% of the acrosome-reacted sperm population no longer demonstrated binding of the PH-20 antibody on the posterior head, but showed binding instead on the inner acrosomal membrane. This redistribution of the PH-20 antigen also resulted from the migration of pre-existing surface molecules, but did not appear to require energy. The migration of PH-20 antigen was a selective process; other antigens localized to the posterior head region did not leave the posterior head after the

  4. Challenges in cryopreserving endangered mammal spermatozoa: morphology and the value of acrosomal integrity as markers of cryo-survival.

    PubMed

    Pukazhenthi, Budhan; Santymire, Rachel; Crosier, Adrienne; Howard, JoGayle; Wildt, David E

    2007-01-01

    The science of cryobiology is essential to the effective, practical use of semen for assisted breeding to help manage small populations of rare wildlife species. In this review, we describe challenges associated with cryopreserving gametes from wild fauna. Based on more than 25 years of experience across a diversity of mammals, it appears that the primary driving force dictating cryo-survival of a spermatozoon is its initial pre-freeze quality and morphology, especially having a morphologically normal, intact acrosome. This assertion is supported through extensive studies of three animal groups that routinely ejaculate semen containing (1) normal sperm/acrosomal quality (examples, Eld's deer, Cervus eldi and giant panda, Ailuropoda melanoleuca), (2) normal acrosomal quality, but from teratospermic donors (>70% pleiomorphic sperm; cheetah, Acinonyx jubatus and black-footed ferret, Mustela nigripes) and (3) abnormal acrosomal quality and general teratospermia (clouded leopard, Neofelis nebulosa). Data revealed that species producing high quality sperm with > 70% normal, intact acrosomes were best able to survive cryopreservation (-80% intact acrosomes post-thaw). Species that were teratospermic, but with high proportions of intact acrosomes (72 to 88%) in ejaculates varied significantly (4 to 55% intact acrosomes post-thaw) in sperm survival to freeze-thawing. Spermatozoa from the clouded leopard (that was both teratospermic while producing only 11% normal acrosomes in fresh semen) failed to survive cryopreservation despite using an array of conventional and unconventional freezing approaches. These observations (combined with zona penetration assays and artificial insemination results) suggest that proportions of malformed sperm and especially initial structural integrity of the acrosome are more important predictors of sperm survivability post-thaw than initial sperm motility scores.

  5. RIM, Munc13, and Rab3A Interplay in Acrosomal Exocytosis

    PubMed Central

    Bello, Oscar D.; Zanetti, M. Natalia; Mayorga, Luis S.; Michaut, Marcela A.

    2012-01-01

    Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have a central role in membrane docking. PMID:22248876

  6. Distribution and dynamics of mouse sperm surface galactosyltransferase: implications for mammalian fertilization.

    PubMed

    Cardullo, R A; Wolf, D E

    1995-08-08

    It has been proposed that a mouse sperm surface beta-1,4-galactosyltransferase functions as a receptor for the zona pellucida during fertilization. In this paper we used two monovalent fluorescent probes specific for galactosyltransferase: a trinitrophenylated derivative of UDP-galactose and rhodaminated alpha-lactalbumin. We found that galactosyltransferase was initially present over the posterior head of acrosome-intact sperm but became progressively localized to the plasma membrane overlying the acrosomal region after it was cross-linked with an anti-galactosyltransferase polyclonal antibody. Labeled mouse sperm that were treated with the calcium ionophore A23187 revealed that galactosyltransferase remained on the posterior head after acrosomal exocytosis. However, if galactosyltransferase was first cross-linked and redistributed with antibody and then acrosome reacted with A23187, all head fluorescence was lost. In addition, although anti-galactosyltransferase antibody induced a surface redistribution, it did not, by itself, lead to the release of acrosin, the endpoint of the acrosome reaction. Finally, using the technique of fluorescence recovery after photobleaching, we found that, in the absence of bivalent antibody, mouse sperm surface galactosyltransferase exhibited 40-50% recovery with a high diffusion coefficient on the anterior head (5-8 x 10(-9) cm2/s) approximately 2 times greater than on the posterior head (2-4 x 10(-9) cm2/s). When galactosyltransferase was cross-linked and redistributed to the anterior head using the bivalent antibody, the mobile fraction decreased to 20-30% with no significant change in the diffusion coefficient.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

    PubMed

    Serafini, R; Longobardi, V; Spadetta, M; Neri, D; Ariota, B; Gasparrini, B; Di Palo, R

    2014-02-01

    Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.

  8. Capacitation inducers act through diverse intracellular mechanisms in cryopreserved bovine sperm.

    PubMed

    Breininger, E; Cetica, P D; Beconi, M T

    2010-10-01

    The effect of various capacitation inducers, i.e. heparin, superoxide anion, bicarbonate, adenosine, and caffeine, and their role in intracellular mechanisms involved in capacitation, were studied in cryopreserved bovine sperm. Capacitation was determined by epifluorescence chlortetracycline, protein tyrosine phosphorylation, and the ability of capacitated sperm to undergo an acrosome reaction and fertilize in vitro matured oocytes. Participation of membrane adenylate cyclase and protein kinases (protein kinase A, protein kinase C, and protein tyrosine kinase) was evaluated indirectly (with specific inhibitors). Involvement of reactive oxygen species (ROS) was determined with scavengers of superoxide anion, hydrogen peroxide, or nitric oxide. Percentages of capacitated (27-29%) and acrosome-reacted sperm (23-26%) did not differ (P > 0.05) among various capacitation inducers. Significantly higher rates of IVF were obtained with heparin (43%) or bicarbonate plus caffeine (45%), when compared with control samples (17%). Adding the membrane adenylate cyclase inhibitor diminished capacitation rates with heparin (8%) or adenosine (10%). There was differential protein kinase participation in response to inducers; protein kinase inhibitors diminished cleavage rates in heparin-capacitated sperm relative to controls. There were differences between and within the studied inducers in protein tyrosine phosphorylation patterns. We inferred that capacitation in cryopreserved bovine sperm was promoted through diverse pathways. Mechanisms triggered by heparin, or caffeine plus bicarbonate-induced capacitation, involved activation of intracellular pathways to optimize fertilizing capability of cryopreserved bovine sperm.

  9. Superoxide anion production by human spermatozoa as a part of the ionophore-induced acrosome reaction process.

    PubMed

    Griveau, J F; Renard, P; Le Lannou, D

    1995-04-01

    The involvement of superoxide anion (O2o-) in human sperm capacitation and/or acrosome reaction was investigated. Addition of superoxide dismutase (SOD) to the medium at the beginning of the capacitation process or 15 min before induction of the acrosome reaction, decreased the level of ionophore-induced acrosome reaction. Hyperactivation was unaffected by the presence of SOD during the capacitation process. Addition of calcium ionophore to the sperm suspension increased production of O2o- by the spermatozoa by four to five-fold and induced the acrosome reaction. In the presence of SOD, superoxide anion could not be detected in the medium and the rate of induced-acrosome reaction was decreased greatly. The presence of an inhibitor of protein kinase C inhibited the production of O2o- in the medium and reduced the induced-acrosome reaction. The production of O2o- and the acrosome reaction were also increased by exposure of spermatozoa to 12-myristate 13-acetate phorbol ester, a specific activator of protein kinase C. While the level of spontaneous acrosome reaction was not increased by the direct addition of O2o- to the medium, its presence induced the release of unesterified fatty acids from membrane phospholipids. These findings suggest that the production of O2o- by spermatozoa could be involved in the ionophore-induced acrosome reaction, possibly through the de-esterification of membrane phospholipids. However, this production of superoxide anion is not sufficient on its own to induce the acrosome reaction.

  10. Intracellular Ca(2+)-Mg(2+)-ATPase regulates calcium influx and acrosomal exocytosis in bull and ram spermatozoa.

    PubMed

    Dragileva, E; Rubinstein, S; Breitbart, H

    1999-11-01

    Calcium influx is required for the mammalian sperm acrosome reaction (AR), an exocytotic event occurring in the sperm head prior to fertilization. We show here that thapsigargin, a highly specific inhibitor of the microsomal Ca(2+)-Mg(2+)-ATPase (Ca(2+) pump), can initiate acrosomal exocytosis in capacitated bovine and ram spermatozoa. Initiation of acrosomal exocytosis by thapsigargin requires an influx of Ca(2+), since incubation of cells in the absence of added Ca(2+) or in the presence of the calcium channel blocker, La(3+), completely inhibited thapsigargin-induced acrosomal exocytosis. ATP-Dependent calcium accumulation into nonmitochondrial stores was detected in permeabilized sperm in the presence of ATP and mitochondrial uncoupler. This activity was inhibited by thapsigargin. Thapsigargin elevated the intracellular Ca(2+) concentration ([Ca(2+)](i)), and this increase was inhibited when extracellular Ca(2+) was chelated by EGTA, indicating that this rise in Ca(2+) is derived from the external medium. This rise of [Ca(2+)](i) took place first in the head and later in the midpiece of the spermatozoon. However, immunostaining using a polyclonal antibody directed against the purified inositol 1,4,5-tris-phosphate receptor (IP(3)-R) identified specific staining in the acrosome region, in the postacrosome, and along the tail, but not in the midpiece region. No staining in the acrosome region was observed in sperm without acrosome, indicating that the acrosome cap was stained in intact sperm. The presence of IP(3)-R in the anterior acrosomal region as well as the induction, by thapsigargin, of intracellular Ca(2+) elevation in the acrosomal region and acrosomal exocytosis, implicates the acrosome as a potential cellular Ca(2+) store. We suggest here that the cytosolic Ca(2+) is actively transported into the acrosome by an ATP-dependent, thapsigargin-sensitive Ca(2+) pump and that the accumulated Ca(2+) is released from the acrosome via an IP(3)-gated calcium

  11. Effect of estrogens on boar sperm capacitation in vitro

    PubMed Central

    2010-01-01

    Background Mammalian sperm must undergo a series of controlled molecular processes in the female reproductive tract called capacitation before they are capable of penetrating and fertilizing the egg. Capacitation, as a complex biological process, is influenced by many molecular factors, among which steroidal hormone estrogens play their role. Estrogens, present in a high concentration in the female reproductive tract are generally considered as primarily female hormones. However, there is increasing evidence of their important impact on male reproductive parameters. The purpose of this study is to investigate the effect of three natural estrogens such as estrone (E1), 17beta-estradiol (E2) and estriol (E3) as well as the synthetical one, 17alpha-ethynylestradiol (EE2) on boar sperm capacitation in vitro. Methods Boar sperm were capacitated in vitro in presence of estrogens. Capacitation progress in control and experimental samples was analyzed by flow cytometry with the anti-acrosin monoclonal antibody (ACR.2) at selected times of incubation. Sperm samples were analyzed at 120 min of capacitation by CTC (chlortetracycline) assay, immunocytochemistry and flow cytometry with anti-acrosin ACR.2 antibody. Furthermore, sperm samples and capacitating media were analyzed by immunocytochemistry, ELISA with the ACR.2 antibody, and the acrosin activity assay after induced acrosomal reaction (AR). Results Estrogens stimulate sperm capacitation of boar sperm collected from different individuals. The stimulatory effect depends on capacitation time and is highly influenced by differences in the response to estrogens such as E2 by individual animals. Individual estrogens have relatively same effect on capacitation progress. In the boar samples with high estrogen responsiveness, estrogens stimulate the capacitation progress in a concentration-dependent manner. Furthermore, estrogens significantly increase the number of acrosome-reacted sperm after zona pellucida- induced acrosomal

  12. [Progress in researches on A-kinase anchor proteins in sperm].

    PubMed

    Li, Yang; Liu, Jihong

    2004-09-01

    This article reviews the advances in the studies of A-kinase anchor proteins (AKAPs) in sperm, including their classification, structure and mechanism. The influence of AKAPs that are involved on sperm mobility and acrosome reaction is emphasized. We hope it could play a directive role in the studies of AKAPs that are involved in regulating sperm mobility and acrosome reaction.

  13. Reacting to "Reacting"

    ERIC Educational Resources Information Center

    Houle, Amanda

    2006-01-01

    This article describes the author's experiences as a student participating in a general education program called "Reacting to the Past," in which college students play elaborate games set at pivotal moments in the past, their roles informed by great texts. She found that the experience of reenacting pivotal historical moments produced an intensely…

  14. Mobile phones affect multiple sperm quality traits: a meta-analysis

    PubMed Central

    Dama, Madhukar Shivajirao

    2013-01-01

    As mobile phone usage is growing rapidly, there is a need for a comprehensive analysis of the literature to inform scientific debates about the adverse effects of mobile phone radiation on sperm quality traits. Therefore, we conducted a meta-analysis of the eligible published research studies on human males of reproductive age. Eleven studies were eligible for this analysis. Based on the meta-analysis, mobile phone use was significantly associated with deterioration in semen quality (Hedges’s g = -0.547; 95% CI: -0.713, -0.382; p < 0.001). The traits particularly affected adversely were sperm concentration, sperm morphology, sperm motility, proportion of non-progressive motile sperm (%), proportion of slow progressive motile sperm (%), and sperm viability. Direct exposure of spermatozoa to mobile phone radiation with in vitro study designs also significantly deteriorated the sperm quality (Hedges’s g = -2.233; 95% CI: -2.758, -1.708; p < 0.001), by reducing straight line velocity, fast progressive motility, Hypo-osmotic swelling (HOS) test score, major axis (µm), minor axis (µm), total sperm motility, perimeter (µm), area (µm 2), average path velocity, curvilinear velocity, motile spermatozoa, and  acrosome reacted spermatozoa (%). The strength of evidence for the different outcomes varied from very low to very high. The analysis shows that mobile phone use is possibly associated with a number of deleterious effects on the spermatozoa. PMID:24327874

  15. Genetic sperm defects.

    PubMed

    Chenoweth, Peter J

    2005-08-01

    Genetic sperm defects are specific sperm defects, which have been shown to have a genetic mode of transmission. Such genetic linkage, either direct or indirect, has been associated with a number of sperm defects in different species, with this number increasing with improved diagnostic capabilities. A number of sperm defects, which have proven or suspected genetic modes of transmission are discussed herein, with particular emphasis on cattle. These include: 1. Acrosome defects (knobbed, ruffled and incomplete); 2. Head defects (abnormal condensation, decapitated, round head, rolled head, nuclear crest); 3. Midpiece abnormalities ("Dag" defect, "corkscrew" defect, "pseudo-droplet" defect); 4. Tail defects ("tail stump" defect, primary ciliary dyskinesia).

  16. Effect of NGF on the motility and acrosome reaction of golden hamster spermatozoa in vitro.

    PubMed

    Jin, WanZhu; Tanaka, Akane; Watanabe, Gen; Matsuda, Hiroshi; Taya, Kazuyoshi

    2010-08-01

    Motility and fertilizing ability are known to be two important physiological attributes of a mature sperm, yet the mechanism by which spermatozoa mature and become motile remains largely unknown. It has been shown that nerve growth factor (NGF) is a protein essential for the development, maintenance and survival of the peripheral and central nervous systems. However, the presence of high levels of NGF protein and mRNA do not correlate with the innervations by NGF sensitive fibers in tissues such as the testis, prostate and seminal vesicles. These observations have shifted the attention of research to the role of NGF outside of the nervous system. Here, we demonstrate that NGF and its receptors TrkA and p75 are widely expressed in the testis, accessory reproductive organ, and the epididymal sperms. We also show that NGF stimulates two important aspects of sperm functions, motility and the acrosome reaction, in a time- and dose-dependent manner. NGF activated the sperm cell acrosome reaction, while addition of inhibitors specific for MAPK kinase significantly blocked the sperm acrosome reaction. Taken together, our findings suggest that NGF plays an integral role in sperm motility and the acrosome reaction through, at least in part, the MAPK signalling pathway.

  17. Heterologous in vitro fertilization and sperm capacitation in an endangered African antelope, the scimitar-horned oryx (Oryx dammah).

    PubMed

    Roth, T L; Weiss, R B; Buff, J L; Bush, L M; Wildt, D E; Bush, M

    1998-02-01

    Scimitar-horned oryx sperm function was studied using protocols developed for domestic cattle. Objectives were to assess sperm 1) viability and motility in vitro over time, 2) capacitation in heparin- or calcium-supplemented medium, and 3) function in an in vitro fertilization system using heterologous (domestic cow) oocytes. Seminal aliquots were washed, and sperm were resuspended in 1) Talp with 5% fetal calf serum (TALP), 2) TALP + 10 microM heparin, 3) TALP + 20 microM heparin, and 4) TALP + 10 mM CaCl. At 0, 3, and 6 h, aliquots were evaluated for sperm motility, viability (using Hoechst 33258), and ability to acrosome-react when exposed to lysophosphatidylcholine (LC). Sperm function was assessed by evaluating fertilization and embryo development after coculture of in vitro-matured domestic cow oocytes with oryx sperm. Overall mean percentages of motile and viable sperm remained high at 6 h (> 60% and > 70%, respectively). Fewer (p < 0.05) sperm incubated in TALP + 10 microM heparin for 6 h contained intact acrosomes after exposure to LC, but there were no differences between LC and control samples after incubation in TALP without heparin. LC-treated sperm in TALP + 10 mM CaCl contained fewer (p < 0.05) intact acrosomes at 3 and 6 h (52.6% and 31.2%, respectively) than paired controls (83.6% and 70.0%, respectively). Oryx sperm from all males were capable of fertilizing cow oocytes (range 17 of 26 [65.4%] to 25 of 26 [96.2%]). Of the 55 2-cell embryos produced, 34 (61.8%) developed to > or = 8 cells. Of the 24 uncleaved oocytes, 7 (29.2%) were polyspermic. These data demonstrate that processed sperm from the endangered scimitar-horned oryx remain vigorous in vitro for at least 6 h. Capacitation can be induced using cattle sperm-processing techniques, with sperm appearing most responsive to elevated CaCl concentrations. Most interesting was the successful production and development of hybrid embryos after coincubation of oryx sperm with cow oocytes, suggesting

  18. Progesterone Accelerates the Completion of Sperm Capacitation and Activates CatSper Channel in Spermatozoa from the Rhesus Macaque1

    PubMed Central

    Sumigama, Shiho; Mansell, Steven; Miller, Melissa; Lishko, Polina V.; Cherr, Gary N.; Meyers, Stuart A.; Tollner, Theodore

    2015-01-01

    During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 μM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa “priming” as they move into a gradient of progesterone in search for the oocyte. PMID:26490839

  19. Progesterone Accelerates the Completion of Sperm Capacitation and Activates CatSper Channel in Spermatozoa from the Rhesus Macaque.

    PubMed

    Sumigama, Shiho; Mansell, Steven; Miller, Melissa; Lishko, Polina V; Cherr, Gary N; Meyers, Stuart A; Tollner, Theodore

    2015-12-01

    During transit through the female reproductive tract, mammalian spermatozoa are exposed to increasing concentrations of progesterone (P4) released by the cumulus oophorus. P4 triggers massive calcium influx into human sperm through activation of the sperm-specific calcium channel CatSper. These properties of human spermatozoa are thought to be unique since CatSper is not progesterone sensitive in rodent sperm. Here, by performing patch clamp recording from spermatozoa from rhesus macaque for the first time, we report that they express P4-sensitive CatSper channel identically to human sperm and react to P4 by inducing responsiveness to zona pellucida, unlike human sperm, which respond directly to P4. We have also determined the physiologic levels of P4 capable of inducing capacitation-associated changes in macaque sperm. Progesterone (1 μM) induced up to a 3-fold increase in the percentage of sperm undergoing the zona pellucida-induced acrosome reaction with the lowest threshold as low as 10 nM of P4. Submicromolar levels of P4 induced a dose-dependent increase in curvilinear velocity and lateral head displacement, while sperm protein tyrosine phosphorylation was not altered. Macaque spermatozoa exposed to 10 μM of P4 developed fully hyperactivated motility. Similar to human sperm, on approaching cumulus mass and binding to zona pellucida, macaque spermatozoa display hyperactivation and undergo an acrosome reaction that coincides with the rise in the sperm intracellular calcium. Taken together, these data indicate that P4 accelerates the completion of capacitation and provides evidence of spermatozoa "priming" as they move into a gradient of progesterone in search for the oocyte.

  20. Cofilin is correlated with sperm quality and influences sperm fertilizing capacity in humans.

    PubMed

    Chen, S M; Chen, X M; Lu, Y L; Liu, B; Jiang, M; Ma, Y X

    2016-11-01

    Spermatozoa should undergo a series of biochemical modifications in female reproduction tract, which is collectively called sperm capacitation. The capacitated spermatozoa can bind to the egg zona pellucida, resulting in the occurrence of acrosome reaction which enabled spermatozoa penetrate into the egg. The formation of actin plays an important role in these processes. Actin polymerized during sperm capacitation, but the polymers dispersed before acrosome reaction. In this study, we take our focus on actin-binding protein, cofilin. Our results showed that the % and intensity of sperm expressing cofilin in normal sperm were significantly higher than in abnormal sperm, and the sperm expressing cofilin was correlated with sperm quality. Furthermore, treatment with anti-cofilin antibody increased the percentage of sperm capacitation and inhibited progesterone- or A23187- induced acrosome reaction in a dose-dependent manner. The presence of 100 ng/mL anti-cofilin antibodies markedly blocked the sperm penetration of zona-free hamster eggs. Besides, immunofluorescence results revealed that cofilin was colocalized with F-actin in the midpiece of spermatozoa; however, phospho-cofilin was expressed in the tail rather than in the midpiece of spermatozoa, which was not colocalized with F-actin in spermatozoa. Moreover, western blot revealed that phospho-cofilin increased in sperm capacitation, and the total cofilin and cofilin in insoluble fraction increased in acrosome reaction; immunofluorescence results showed that the amount of cofilin in acrosome increased in sperm capacitation. In conclusion, our study revealed that cofilin expression in human sperm is correlated with sperm quality and the alterations of cofilin and phospho-cofilin in fertilization affects sperm capacitation, acrosome reaction, and spermatozoa-oocyte fusion.

  1. Acrosomo-nuclear syndrome in canine sperm.

    PubMed

    Hrudka, F

    1983-01-01

    An acrosomo-nuclear syndrome in sperm of an infertile semicryptorchid dog is described. Based on an EM study of developing and mature sperm the syndrome is defined by simultaneous occurrence of these symptoms: 1) intranuclear inclusions of acrosomal origin, 2) maldifferentiated apical segment of acrosome, 3) intraacrosomal inclusions of Sertoli cell origin, 4) characteristic change of nuclear shape and 5) retained cytoplasmic droplet. The cause of the syndrome and possibility of a transfer of somatic factors are discussed.

  2. Activation of proacrosin accompanies upregulation of sp32 protein tyrosine phosphorylation in pig sperm.

    PubMed

    Sun, P L; Yang, L X; Cui, J-J; Tian, Y; Liu, Y; Jin, Y

    2013-12-11

    This study investigated the relationship between acrosin activation and pig sperm proacrosin binding protein (sp32) phosphorylation levels. Differently processed pig spermatozoa (fresh semen sperm, capacitation sperm, acrosome reaction sperm, capacitation-like sperm, and thawed sperm) were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis. The fresh semen and capacitation sperm groups both produced proacrosin protein bands of 55 kDa; however, the result of the fresh semen sperm group was clearer than that of the capacitation sperm group. The thawed sperm group showed a shallow strip at 55 kDa. The capacitation and acrosome reaction sperm groups produced obvious proacrosin protein bands at 35 kDa, and the strips of the capacitation sperm group were again clearer. A faint band was visible at 32 kDa in the acrosome reaction sperm group. The capacitation, thawed, and acrosome reaction sperm groups showed significant strips in sp32, and the bands of the acrosome reaction sperm group were shallower than those of the 2 other groups. The capacitation and thawed sperm groups produced significant strips at 40 kDa, and the capacitation sperm group produced an additional strip at 55 kDa. In conclusion, sp32 phosphorylation levels can promote proacrosin activation into the active acrosin.

  3. Phosphatidylinositol 3-kinase pathway regulates sperm viability but not capacitation on boar spermatozoa.

    PubMed

    Aparicio, I M; Bragado, M J; Gil, M C; Garcia-Herreros, M; Gonzalez-Fernandez, L; Tapia, J A; Garcia-Marin, L J

    2007-08-01

    Phosphatidylinositol 3-kinase (PI3-K) plays an important role in cell survival in somatic cells and recent data pointed out a role for this kinase in sperm capacitation and acrosome reaction (AR). This study was undertaken to evaluate the role of PI3-K pathway on porcine spermatozoa capacitation, AR, and viability using two unrelated PI3-K inhibitors, LY294002 and wortmannin. In boar spermatozoa, we have identified the presence of PDK1, PKB/Akt, and PTEN, three of the main key components of the PI3-K pathway. Incubation of boar sperm in a capacitating medium (TCM) caused a significant increase in the percentage of capacitated (25 +/- 2 to 34 +/- 1% P < 0.05, n = 6) and acrosome reacted (1 +/- 1 to 11 +/- 1% P < 0.01, n = 6) spermatozoa compared with sperm in basal medium (TBM). Inhibition of PI3-K did affect neither the capacitation status nor AR nor protein p32 tyrosine phosphorylation of boar spermatozoa incubated in TBM or TCM. Boar sperm viability in TBM was significantly decreased by 40 and 20% after pretreatment with LY294002 or wortmannin, respectively. Similar results were observed after incubation of boar spermatozoa in TCM. Treatment of boar spermatozoa with the analog of cAMP, 8Br-cAMP significantly prevented the reduction on sperm viability. Our results provide evidence for an important role of the PI3-K pathway in the regulation of boar sperm viability and suggests that other signaling pathways different from PI3-K must be activated downstream of cAMP to contribute to regulation of sperm viability. Finally, in our conditions the PI3-K pathway seems not related with boar sperm capacitation or AR.

  4. Oviductosome-Sperm Membrane Interaction in Cargo Delivery: DETECTION OF FUSION AND UNDERLYING MOLECULAR PLAYERS USING THREE-DIMENSIONAL SUPER-RESOLUTION STRUCTURED ILLUMINATION MICROSCOPY (SR-SIM).

    PubMed

    Al-Dossary, Amal A; Bathala, Pradeepthi; Caplan, Jeffrey L; Martin-DeLeon, Patricia A

    2015-07-17

    Oviductosomes ((OVS), exosomes/microvesicles), which deliver the Ca(2+) efflux pump, plasma membrane Ca(2+)ATPase 4 (PMCA4), to sperm are likely to play an important role in sperm fertilizing ability (Al-Dossary, A. A., Strehler, E. E., and Martin-DeLeon, P. A. (2013) PloS one 8, e80181). It is unknown how exosomes/microvesicles deliver transmembrane proteins such as PMCA4 to sperm. Here we define a novel experimental approach for the assessment of the interaction of OVS with sperm at a nanoscale level, using a lipophilic dye (FM4-64FX) and three-dimensional SR/SIM, which has an 8-fold increase in volumetric resolution, compared with conventional confocal microscopy. Coincubation assays detected fusion of prelabeled OVS with sperm, primarily over the head and midpiece. Immunofluorescence revealed oviductosomal delivery of PMCA4a to WT and Pmca4 KO sperm, and also endogenous PMCA4a on the inner acrosomal membrane. Fusion was confirmed by transmission immunoelectron microscopy, showing immunogold particles in OVS, and fusion stalks on sperm membrane. Immunofluorescence colocalized OVS with the αv integrin subunit which, along with CD9, resides primarily on the sperm head and midpiece. In capacitated and acrosome reacted sperm, fusion was significantly (p < 0.001) inhibited by blocking integrin/ligand interactions via antibodies, exogenous ligands (vitronectin and fibronectin), and their RGD recognition motif. Our results provide evidence that receptor/ligand interactions, involving αvβ3 and α5β1integrins on sperm and OVS, facilitate fusion of OVS in the delivery of transmembrane proteins to sperm. The mechanism uncovered is likely to be also involved in cargo delivery of prostasomes, epididymosomes, and uterosomes.

  5. Decoding mechanisms of loss of fertilization ability of cryopreserved mouse sperm

    NASA Astrophysics Data System (ADS)

    Gray, Jeffrey Earl

    Cryopreservation of mouse sperm is an important technology for management of biomedical research resources. Dramatic progress has been made recently in the development of protocols that combat mouse-strain specific reduction of IVF after cryopreservation. Equal emphasis, however, has not been placed on investigating the biological mechanisms underlying these improvements to IVF. This dissertation broadly investigates the basic question of how mouse-strain specific reduction of IVF occurs after cryopreservation, and how recently developed protocols prevent this process. My research investigated the effects of antioxidants, the cholesterol-acceptor CD, reduced calcium media, and TYH capacitation media on sperm function and oxidative stress after cryopreservation in a variety of mouse strains. I found that reduced IVF was associated with loss of capacitation-dependent sperm function in three strains, B6/J, B6/N, and 129X1, and CD improved sperm function and IVF in all three strains. These findings suggest that cryopreservation inhibits cholesterol efflux resulting in reduced IVF of many mouse strains. I also found that cryopreservation induces uniquely high production of mitochondrial H2O2 by B6/J sperm. H2O2 present in other cellular compartments of B6/J sperm was not elevated compared to other strains. High levels of mitochondrial H2O2 were associated with lipid peroxidation of the sperm head and inability to acrosome react. Antioxidants reduced mitochondrial H2O2 production, decreased sperm head lipid peroxidation, and improved acrosome reaction. The cryopreservation-induced increase in mitochondrial H2O2 production of B6/J and B6129XF1 sperm was associated with elevation of intracellular calcium after cryopreservation and dependent on mitochondrial metabolic substrates. Reducing intracellular calcium levels or removing mitochondrial metabolic substrates decreased mitochondrial H2O2 production and increased IVF rates of cryopreserved B6/J sperm. Many of the strains

  6. Effect of egg yolk medium on the acrosome reaction of human spermatozoa.

    PubMed

    Bielfeld, P; Jeyendran, R S; Holmgren, W J; Zaneveld, L J

    1990-01-01

    Preincubation of human spermatozoa in an egg yolk medium (TESTY) at 5 C, followed by washing at 37 C by centrifugation and resuspension in a standard medium (BWW), enhanced the percentage of spermatozoa that underwent the acrosome reaction and increased sperm penetration into zona-free hamster oocytes, as compared with BWW treatment only. The difference in the occurrence of the acrosome reaction between the two treatment protocols was present whether the spermatozoa were incubated for 3 or for 18 h. The increase in acrosome reaction occurred only when spermatozoa were washed after TESTY treatment. Washing at 5 C was not as effective as washing at 37 C. No increased loss of acrosomes was observed when BWW-treated spermatozoa were subjected to the washing procedure. Ionophore A23187 stimulated the acrosome reaction of BWW-treated but not of TESTY-treated spermatozoa, whether or not they were washed before ionophore treatment. In the absence of egg yolk, the medium (TEST) caused only a small enhancement in the acrosome reaction as compared with BWW, but an increase occurred upon addition of ionophore A23187. We conclude that treatment with TESTY enhances the capacitation/acrosome reaction of human spermatozoa and that the removal of egg yolk after incubation, as well as the temperature shock, contribute to this effect.

  7. Destabilization of acrosome and elastase influence mediate the release of secretory phospholipase A2 from human spermatozoa.

    PubMed

    Lessig, Jacqueline; Reibetanz, Uta; Arnhold, Jurgen; Glander, Hans-Jurgen

    2008-11-01

    To determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes. Acrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used. Although sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality. The ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality. (c) 2008, Asian Journal of Andrology, SIMM and SJTU. All rights reserved.

  8. Morphological and acrosomal changes of canine spermatozoa during epididymal transit

    PubMed Central

    2013-01-01

    Background During epididymal transit, functional and structural modifications leading to full maturation enable male gametes to reach, recognize and fertilize the oocytes. In dogs, little is known on the modifications of spermatozoa during the passage in the epididymis. The aim of this study was to describe the motility, morphology and acrosomal patterns of canine spermatozoa retrieved from the epididymis caput, corpus and cauda. Results After the dilution required for the collection of epididymal content, sperm motility was significantly higher (P <0.0001) in the cauda compared to corpus and caput. Proportions of spermatozoa with normal morphology were significantly higher in corpus (P =0.02) and cauda (P <0.0001) compared to caput. Overall morphological abnormalities of the head and neck/midpiece were similar in the three different epididymal regions. A significantly increased prevalence of tail defects, mainly represented by single bent tails, was observed in the corpus compared to caput (P <0.0001) and cauda (P =0.006). Numbers of immature sperm with cytoplasmic droplets decreased from the proximal to the distal region of the epididymis. Particularly, proximal cytoplasmic droplets were more frequently found in spermatozoa collected from the caput epididymis than in the corpus (P <0.0001) and in the cauda (P <0.0001), whereas the occurrence of distal cytoplasmic droplets was higher in the corpus than in the caput (P =0.0003) and in the cauda (P <0.05). Significantly higher proportions of spermatozoa with intact acrosomes were retrieved from the cauda epididymis than from the caput (P =0.03) and the corpus (P =0.008). This difference was mainly due to a lower proportion of spermatozoa with abnormal acrosomes (mainly swollen acrosomes) rather than with absent acrosomes. Conclusions Canine spermatozoa undergo several modifications in the epididymis. The acquisition of progressive motility, migration of the cytoplasmic droplet and acrosomal reshaping lead to mature

  9. Use of antioxidants reduce lipid peroxidation and improve quality of crossbred ram sperm during its cryopreservation.

    PubMed

    Banday, Mohamad Naiem; Lone, Farooz Ahmad; Rasool, Fabiha; Rashid, Muzamil; Shikari, Arif

    2017-02-01

    Ram sperm are subjected to extreme oxidative stress during their preservation at -196 °C resulting in reduced quality at post thaw. Therefore, the main objective of this study was to evaluate the effect of antioxidants taurine, quercetin and reduced glutathione on the post thaw quality of crossbred ram sperm. A total of twenty four ejaculates from six crossbred rams were collected and extended with tris-based extender with no antioxidant (Control), with taurine (40 mM), quercetin (5 μg/ml) and reduced glutathione (5 mM). The post thaw sperm quality was determined by percent sperm motility, live sperm count, intact acrosome and hypo-osmotic swelling test (HOST) reacted spermatozoa and lipid peroxidation was measured in terms of malondialdehyde (MDA) level both in seminal plasma and sperm cell. At post thaw, percent sperm motility and live sperm count were significantly (p < 0.05) higher for taurine than control and reduced glutathione but did not differ significantly (p > 0.05) from quercetin. The percent HOST reacted spermatozoa were significantly higher for taurine than control, quercetin and reduced glutathione. Seminal plasma MDA level was significantly (p < 0.05) lower for taurine than control and non-significantly lower than quercetin and reduced glutathione. However, spermatic MDA level did not differ significantly (p > 0.05) among the control and antioxidants. In conclusion, taurine at 40 mM reduced lipid peroxidation and improved post thaw sperm quality of cryopreserved crossbred ram semen. Further, transportation time of semen samples in an ice chest at 4-5 °C may be included as a part of equilibration period, when collection shed and frozen semen unit are located at a distance. Copyright © 2016 Elsevier Inc. All rights reserved.

  10. Acidic hyaluronidase activity is present in mouse sperm and is reduced in the absence of SPAM1: Evidence for a Role for Hyaluronidase 3 in mouse and human sperm

    PubMed Central

    Reese, Kristen L.; Aravindan, Rolands G.; Griffiths, Genevieve S.; Shao, Minghai; Wang, Yipei; Galileo, Deni S.; Atmuri, Vasantha; Triggs-Raine, Barbara L.; Martin-DeLeon, Patricia A.

    2010-01-01

    The molecular mechanisms underlying sperm penetration of the physical barriers surrounding the oocyte have not been completely delineated. Although neutral-active or “reproductive” hyaluronidases (hyases), exemplified by Sperm Adhesion Molecule 1 (SPAM1), are thought to be responsible for hyaluronan digestion in the egg vestments and for sperm-zona binding, their roles in mouse sperm have been recently questioned. Here we report that acidic “somatic” Hyaluronidase 3 (HYAL3) exists in two isoforms in human (~47 kDa, ~55 kDa) and mouse (~44, ~47kDa) sperm where it resides on the plasma membrane over the head and midpiece. Mouse isoforms are differentially distributed in the soluble (SAP), membrane (MBP), and acrosome-reacted (AR) fraction where they are most abundant. Comparisons of zymography of Hyal3 null and wild-type (WT) AR and MBP fractions show significant HYAL3 activity at pH 3 and 4, and less at 7. At pH 4, a second acid-active hyase band at ~57 kDa is present in the AR fraction. HYAL3 activity was confirmed using immunoprecipitated HYAL3 and spectrophotometry. In total proteins, hyase activity was higher at pH 6 than at 4 where Spam1 nulls had significantly (P<0.01) diminished activity, indicating that murine SPAM1 has acidic activity. Although fully fertile, Hyal3 null sperm showed delayed cumulus penetration and reduced acrosomal exocytosis. HYAL3, similar to SPAM1 with which it shares 74.6% structural similarity, exists in epididymal tissue/fluid from which it is acquired by caudal mouse sperm in vitro. Our results indicate for the first time the concerted activity of both neutral- and acid-active hyaluronidases in sperm. PMID:20586096

  11. Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity.

    PubMed

    Hermosell, Ignacio G; Laskemoen, Terje; Rowe, Melissah; Møller, Anders P; Mousseau, Timothy A; Albrecht, Tomás; Lifjeld, Jan T

    2013-10-23

    Interspecific variation in sperm size is enigmatic, but generally assumed to reflect species-specific trade-offs in selection pressures. Among passerine birds, sperm length varies sevenfold, and sperm competition risk seems to drive the evolution of longer sperm. However, little is known about factors favouring short sperm or constraining the evolution of longer sperm. Here, we report a comparative analysis of sperm head abnormalities among 11 species of passerine bird in Chernobyl, presumably resulting from chronic irradiation following the 1986 accident. Frequencies of sperm abnormalities varied between 15.7 and 77.3% among species, more than fourfold higher than in uncontaminated areas. Nonetheless, species ranked similarly in sperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome damage consistent with premature acrosome reaction. Sperm length, but not sperm competition risk explained variation in sperm damage among species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm length and sperm integrity affecting sperm evolution in passerine birds.

  12. Patterns of sperm damage in Chernobyl passerine birds suggest a trade-off between sperm length and integrity

    PubMed Central

    Hermosell, Ignacio G.; Laskemoen, Terje; Rowe, Melissah; Møller, Anders P.; Mousseau, Timothy A.; Albrecht, Tomáš; Lifjeld, Jan T.

    2013-01-01

    Interspecific variation in sperm size is enigmatic, but generally assumed to reflect species-specific trade-offs in selection pressures. Among passerine birds, sperm length varies sevenfold, and sperm competition risk seems to drive the evolution of longer sperm. However, little is known about factors favouring short sperm or constraining the evolution of longer sperm. Here, we report a comparative analysis of sperm head abnormalities among 11 species of passerine bird in Chernobyl, presumably resulting from chronic irradiation following the 1986 accident. Frequencies of sperm abnormalities varied between 15.7 and 77.3% among species, more than fourfold higher than in uncontaminated areas. Nonetheless, species ranked similarly in sperm abnormalities in unpolluted areas as in Chernobyl, pointing to intrinsic factors underlying variation in sperm damage among species. Scanning electron microscopy of abnormal spermatozoa revealed patterns of acrosome damage consistent with premature acrosome reaction. Sperm length, but not sperm competition risk explained variation in sperm damage among species. This suggests that longer spermatozoa are more susceptible to premature acrosome reaction. Therefore, we hypothesize a trade-off between sperm length and sperm integrity affecting sperm evolution in passerine birds. PMID:24088561

  13. Canine sperm vitrification with sucrose: effect on sperm function.

    PubMed

    Sánchez, R; Risopatrón, J; Schulz, M; Villegas, J; Isachenko, V; Kreinberg, R; Isachenko, E

    2011-08-01

    The ability of sucrose to protect spermatozoa against mitochondrial damage, artificial acrosome reaction and DNA fragmentation during ultra-rapid cryopreservation in canine sperm was investigated. Swim-up selected spermatozoa of second-fraction semen were vitrified with different concentrations of sucrose (0.1, 0.25 and 0.4 m) in proportion 1 : 1 v/v with HTF-BSA 1%. From each group, 30-μl suspensions of cells were dropped directly into liquid nitrogen and stored for at least 24 h. Cells were thawed by submerging the spheres in HTF with 1% BSA at 37 °C. The number of progressively motile spermatozoa was significantly higher in the sucrose 0.25 m + HTF-BSA 1% (42.5 ± 2.3%, P < 0.01) than in HTF only (1.66 ± 0.3%). The same combination of sucrose 0.25 m + HTF-BSA 1% (42.7 ± 1.5%) had a stronger cryoprotective effect on the integrity of mitochondrial membrane potential (P < 0.05) and decreased the DNA fragmentation (2.8 ± 0.5%) as compared with HTF only (1.93 ± 0.6% and 5.6 ± 0.6% respectively). With respect to acrosome-reacted spermatozoa, no significant difference was found between the groups investigated (P > 0.05). It is concluded that sucrose, a nonpermeable cryoprotectant, can effectively preserve important physiological parameters such as mitochondrial membrane potential and DNA integrity during ultra-rapid cryopreservation. © 2011 Blackwell Verlag GmbH.

  14. Lack of acrosome formation in mice lacking a Golgi protein, GOPC

    PubMed Central

    Yao, Ryoji; Ito, Chizuru; Natsume, Yasuko; Sugitani, Yoshinobu; Yamanaka, Hitomi; Kuretake, Shoji; Yanagida, Kaoru; Sato, Akira; Toshimori, Kiyotaka; Noda, Tetsuo

    2002-01-01

    The acrosome is a unique organelle that plays an important role at the site of sperm–zona pellucida binding during the fertilization process, and is lost in globozoospermia, an inherited infertility syndrome in humans. Although the acrosome is known to be derived from the Golgi apparatus, molecular mechanisms underlying acrosome formation are largely unknown. Here we show that Golgi-associated PDZ- and coiled-coil motif-containing protein (GOPC), a recently identified Golgi-associated protein, is predominantly localized at the trans-Golgi region in round spermatids, and male mice in which GOPC has been disrupted are infertile with globozoospermia. The primary defect was the fragmentation of acrosomes in early round spermatids, and abnormal vesicles that failed to fuse to developing acrosomes were apparent. In later stages, nuclear malformation and an abnormal arrangement of mitochondria, which are also characteristic features of human globozoospermia, were observed. Interestingly, intracytoplasmic sperm injection (ICSI) of such malformed sperm into oocytes resulted in cleavage into blastocysts only when injected oocytes were activated. Thus, GOPC provides important clues to understanding the mechanisms underlying spermatogenesis, and the GOPC-deficient mouse may be a unique and valuable model for human globozoospermia. PMID:12149515

  15. Localization of Tektin 1 at both acrosome and flagella of mouse and bull spermatozoa.

    PubMed

    Oiki, Sayoko; Hiyama, Erina; Gotoh, Takafumi; Iida, Hiroshi

    2014-02-01

    Tektins (TEKTs) are constitutive filamentous proteins of microtubules in cilia, flagella, basal bodies, and centrioles. In mammals, five TEKTs (TEKT1, 2, 3, 4, and 5) have been identified in testis and spermatozoa. With the exception of TEKT1, these TEKTs have been reported to be present in spermatozoa with predominant localization at the peri-axoneme structures of flagella, i.e., mitochondria and outer dense fibers. In the present study, we produced an antibody against TEKT1 to examine the localization of TEKT1 in mouse, bull, and rat spermatozoa. By immunoblot analyses and immunofluorescence microscopy, we found TEKT1 to be present in sperm flagella and at the apical region of acrosome cap in spermatozoa of all these species. Acrosome-associated TEKT1 disappeared after in vitro acrosome reaction in mouse spermatozoa. These observations suggest another potential role for TEKT1 as a cytoskeletal element in the sperm head, or as a molecule involved in acrosome-related phenomena, such as acrosome reaction.

  16. Rat sperm immobilisation effects of a protein from Ricinus communis (Linn.): an in vitro comparative study with nonoxynol-9.

    PubMed

    Nithya, R S; Anuja, M M; Rajamanickam, C; Indira, M

    2012-12-01

    Previous study conducted in our department showed that 50% ethanolic extract of the root of Ricinus communis possess reversible antifertility effect and a 62-kDa protein (Rp) from this extract is responsible for the antifertility effects. In this study, we compared the spermicidal effect of this Rp with nonoxynol-9 (N-9) in vitro. The sperm immobilisation studies showed that 100 μg ml(-1) of Rp was able to immobilise the sperms completely within 30 s. Sperm revival test revealed that the spermicidal effect was irreversible. There was also a significant reduction in sperm viability and hypo-osmotic swelling in Rp and N-9 treated groups in comparison with the control. In Rp and N-9 treated groups, the number of acrosome-reacted cells was found to be high and also caused agglutination of the spermatozoa, indicating the loss of intactness of the plasma membrane, which was further supported by the significant reduction in the activity of membrane bound 5'-nucleotidase, acrosomal acrosin. In short, the protein Rp possesses spermicidal activity in vitro and its effects are similar to that of nonoxynol 9. © 2012 Blackwell Verlag GmbH.

  17. Endocannabinoids, sperm functions and energy metabolism.

    PubMed

    Rossato, Marco

    2008-04-16

    Cannabinoids, the main active components of marijuana, have been shown to exert different adverse effects on male reproduction both in vertebrates and invertebrates. The main effects of endocannabinoids, a particular group of endogenously produced cannabinoids, in sperm are the inhibition of motility, capacitation and acrosome reaction, all fundamental processes necessary for oocyte penetration, whose alteration leads to the inhibition of sperm fertilizing ability. These inhibitory effects are mediated by the direct action of endocannabinoids on sperm through the activation of the cannabinoid receptor subtype 1 that has been shown to be expressed in mature sperm. In many different cell types it has been demonstrated that endocannabinoids negatively influence mitochondrial activity. In the present paper it will be briefly reviewed the role of endocannabinoids, on sperm motility, capacitation and acrosome reaction with particular attention on the possible interference of endocannabinoids with sperm mitochondrial activity.

  18. Seipin deficiency increases chromocenter fragmentation and disrupts acrosome formation leading to male infertility.

    PubMed

    El Zowalaty, A E; Baumann, C; Li, R; Chen, W; De La Fuente, R; Ye, X

    2015-07-16

    The Berardinelli-Seip congenital lipodystrophy type 2 (Bscl2, seipin) gene is involved in adipogenesis. Bscl2-/- males were infertile but had normal mating behavior. Both Bscl2-/- cauda epididymis sperm count and sperm motility were ~20×less than control. Bscl2-/- seminiferous tubules had relatively normal presence of spermatogonia and spermatocytes but had reduced spermatids and sperm. Spatiotemporal expression analyses in Bscl2+/+ testes demonstrated prominent Bscl2 transcriptional activity in spermatocytes with a plateau reached around postnatal day 28. Seipin protein localization was most abundant in postmeiotic spermatids, suggesting translational repression of Bscl2 mRNA in spermatocytes. In situ end-labeling plus detected increased spermatid apoptosis in Bscl2-/- testis and annexin V detected increased percentage of positive Bscl2-/- round spermatids compared with control. Immunofluorescence of marker proteins synaptonemal complex proteins 3 and 1 (SYCP3 and SYCP1), and H3K9me3 (histone H3 trimethylated at lysine 9) in germ cell spreads detected normal meiotic chromosome pairing and homologous chromosome synapsis in Bscl2-/- spermatocytes, but significantly increased percentages of round spermatids with chromocenter fragmentation and late spermatids and sperm with chromatin vacuoles, indicating defective chromatin condensation in Bscl2-/- spermatids. Bscl2-/- late spermatids were disorganized within the seminiferous epithelium, despite normal appearance of Sertoli cells detected by vimentin immunofluorescence. Peanut agglutinin staining revealed various abnormalities of acrosomes in Bscl2-/- late spermatids, including the absence, irregular-shaped, and fragmented acrosomes, indicating defective acrosome formation in Bscl2-/- late spermatids, which may affect late spermatid orientation in the seminiferous epithelium. Mitotracker strongly stained the midpiece of control sperm but only very weakly labeled the midpiece of Bscl2-/- sperm, indicating defective

  19. Seipin deficiency increases chromocenter fragmentation and disrupts acrosome formation leading to male infertility

    PubMed Central

    El Zowalaty, A E; Baumann, C; Li, R; Chen, W; De La Fuente, R; Ye, X

    2015-01-01

    The Berardinelli–Seip congenital lipodystrophy type 2 (Bscl2, seipin) gene is involved in adipogenesis. Bscl2−/− males were infertile but had normal mating behavior. Both Bscl2−/− cauda epididymis sperm count and sperm motility were ~20 × less than control. Bscl2−/− seminiferous tubules had relatively normal presence of spermatogonia and spermatocytes but had reduced spermatids and sperm. Spatiotemporal expression analyses in Bscl2+/+ testes demonstrated prominent Bscl2 transcriptional activity in spermatocytes with a plateau reached around postnatal day 28. Seipin protein localization was most abundant in postmeiotic spermatids, suggesting translational repression of Bscl2 mRNA in spermatocytes. In situ end-labeling plus detected increased spermatid apoptosis in Bscl2−/− testis and annexin V detected increased percentage of positive Bscl2−/− round spermatids compared with control. Immunofluorescence of marker proteins synaptonemal complex proteins 3 and 1 (SYCP3 and SYCP1), and H3K9me3 (histone H3 trimethylated at lysine 9) in germ cell spreads detected normal meiotic chromosome pairing and homologous chromosome synapsis in Bscl2−/− spermatocytes, but significantly increased percentages of round spermatids with chromocenter fragmentation and late spermatids and sperm with chromatin vacuoles, indicating defective chromatin condensation in Bscl2−/− spermatids. Bscl2−/− late spermatids were disorganized within the seminiferous epithelium, despite normal appearance of Sertoli cells detected by vimentin immunofluorescence. Peanut agglutinin staining revealed various abnormalities of acrosomes in Bscl2−/− late spermatids, including the absence, irregular-shaped, and fragmented acrosomes, indicating defective acrosome formation in Bscl2−/− late spermatids, which may affect late spermatid orientation in the seminiferous epithelium. Mitotracker strongly stained the midpiece of control sperm but only very weakly labeled the

  20. Participation of phosphofructokinase, malate dehydrogenase and isocitrate dehydrogenase in capacitation and acrosome reaction of boar spermatozoa.

    PubMed

    Breininger, E; Dubois, D; Pereyra, V E; Rodriguez, P C; Satorre, M M; Cetica, P D

    2017-10-01

    The aim of this work was to determine the enzymatic activity of phosphofructokinase (PFK), malate dehydrogenase (MDH) and isocitrate dehydrogenase (IDH) in boar spermatozoa and study their participation in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction. Enzymatic activity of these enzymes was determined spectrophotometrically in extracts of boar spermatozoa. Sperm suspensions were incubated in the presence of bicarbonate (40 mM), a well-known capacitation inducer, or follicular fluid (30%), as an acrosome reaction inducer, and different concentrations of oxoglutarate, oxalomalate and hydroxymalonate, inhibitors of PFK, IDH and MDH, respectively. Capacitation percentages were determined by the fluorescence technique of chlortetracycline (CTC), and true acrosome reaction was determined by trypan blue and differential-interferential contrast, optical microscopy. The activity of PFK in boar spermatozoa enzymatic extracts was 1.70 ± 0.19 U/10(10) spermatozoa, the activity of NAD- and NADP-dependent IDH was 0.111 ± 0.005 U/10(10) and 2.22 ± 0.14 U/10(10) spermatozoa, respectively, and the activity of MDH was 4.24 ± 0.38 U/10(10) spermatozoa. The addition of the specific inhibitors of these enzymes prevented sperm capacitation and decreased sperm motility during capacitation and inhibited the acrosome reaction (AR), without affecting the sperm motility during this process. Our results demonstrate the participation of PFK, IDH and MDH in bicarbonate-induced capacitation and follicular fluid-induced acrosome reaction in boar spermatozoa, contributing to elucidate the mechanisms that produce energy necessary for these processes in porcine spermatozoa. © 2017 Blackwell Verlag GmbH.

  1. [Sequential observation of human sperm penetration into zona pellucida-free hamster oocytes by scanning electron microscopy].

    PubMed

    Tsuiki, A; Hoshi, K; Saito, A; Kyono, K; Hoshiai, H; Suzuki, M; Horiuchi, T

    1983-10-01

    Yanagimachi et al. have shown that zona pellucida-free hamster oocytes can be penetrated by capacitated and acrosome-reacted human spermatozoa, and both pronuclei be formed. Recently, it has been said that this technique may be used to test the fertilizing ability of human sperm for clinical use. We have made scanning electron microscopic studies of the interaction of these heterologous gametes, especially the mechanisms of sperm entry into the oocytes. Our results are as follows: The surface of the oocyte is covered with numerous microvilli, which are found to be evenly and densely distributed on the vitellus surface. The surface of the first polar body already released is smooth and microvilli-free. Most spermatozoa lie flat on the vitellus surface, but a few are oriented perpendicularly to the vitellus surface. Most bound sperm had lost their acrosomal caps, because a ridge exists at the leading edge of the equatorial segment. Many microvilli are shown to participate in sperm-egg contact. Initially most microvilli appeared to grasp and immobilize the anterior tip of the sperm head. But as gamete interaction proceeded, microvilli were overlying the postacrosomal region, and observed adjacent to the plasma membrane of the postacrosomal region. The postacrosomal region is first incorporated into the ooplasma, the anterior tip of sperm head being the last portion to be incorporated. The microvilli of the oolenmal surface where sperm penetrated did not show major changes in size or appearance, and the so-called "incorporation cone" was not observed. Our scanning electron micrographs show that the microvillar portion of the oolenma greatly participates in this heterologous gamate interaction.

  2. Progesterone in mare follicular fluid induces the acrosome reaction in stallion spermatozoa and enhances in vitro binding to the zona pellucida.

    PubMed

    Cheng, F P; Fazeli, A R; Voorhout, W F; Tremoleda, J L; Bevers, M M; Colenbrander, B

    1998-04-01

    The aim of this study was to investigate whether mare follicular fluid (FF) induces the acrosome reaction (AR) in stallion spermatozoa and, if so, to identify the component in FF responsible for it. Furthermore, the effect of this component on sperm-zona binding and the subsequent AR was studied. Pooled FF, aspirated from the preovulatory follicles of mares in oestrous, was used and aliquots of the fluid were treated with charcoal to remove steroids (CFF). Charcoal treatment reduced the progesterone concentration in FF from 153 to < 2 ng/mL. Spermatozoa from fertile stallions collected by a swim-up procedure were preincubated in modified Tyrode's medium for 5 h and then incubated for 30 min at 37 degrees C with either (1) 50% FF + 50% CFF, (2) 50% FF + 50% CFF + 150 ng/mL progesterone, (3) 50% CFF + 150 ng/mL progesterone, (4)150 ng/mL progesterone or (5) modified Tyrode's medium alone. The sperm-hemizona assay was applied: (a) to compare the number of spermatozoa bound to a hemizona in the presence and absence of 1.5, 15 or 150 ng/mL progesterone after 1 h co-incubation of spermatozoa and hemizonae, (b) to compare the incidence of the AR in sperm-hemizona complexes incubated for 1 h in the presence and absence of 1 microgram/mL progesterone. Both spermatozoa in suspension and bound to a hemizona were treated with the supravital dye Ethidium homodimer and fixed. Their plasma membranes were permeabilized, and the outer acrosomal membranes were labelled with FITC-PNA. Viable spermatozoa without the outer acrosomal membrane were considered as physiologically acrosome-reacted. Results showed that (1) FF induced a higher percentage of AR than did CFF or modified Tyrode's medium, (2) addition of 150 ng/mL progesterone to CFF restored 77% of the AR-inducing activity and (3) CFF and modified Tyrode's medium both induced the AR to a similar extent when supplemented with 150 ng/mL progesterone. Neither FF nor progesterone treatment affected sperm viability severely. The

  3. Performance of Rodent Spermatozoa Over Time Is Enhanced by Increased ATP Concentrations: The Role of Sperm Competition.

    PubMed

    Tourmente, Maximiliano; Villar-Moya, Pilar; Varea-Sánchez, María; Luque-Larena, Juan J; Rial, Eduardo; Roldan, Eduardo R S

    2015-09-01

    Sperm viability, acrosome integrity, motility, and swimming velocity are determinants of male fertility and exhibit an extreme degree of variation among closely related species. Many of these sperm parameters are associated with sperm ATP content, which has led to predictions of trade-offs between ATP content and sperm motility and velocity. Selective pressures imposed by sperm competition have been proposed as evolutionary causes of this pattern of diversity in sperm traits. Here, we examine variation in sperm viability, acrosome integrity, motility, swimming velocity, and ATP content over time, among 18 species of closely related muroid rodents, to address the following questions: (a) Do sperm from closely related species vary in ATP content after a period of incubation? (b) Are these differences in ATP levels related to differences in other sperm traits? (c) Are differences in ATP content and sperm performance over time explained by the levels of sperm competition in these species? Our results revealed a high degree of interspecific variability in changes in sperm ATP content, acrosome integrity, sperm motility and swimming velocity over time. Additionally, species with high sperm competition levels were able to maintain higher levels of sperm motility and faster sperm swimming velocity when they were incubated under conditions that support sperm survival. Furthermore, we show that the maintenance of such levels of sperm performance is correlated with the ability of sperm to sustain high concentrations of intracellular ATP over time. Thus, sperm competition may have an important role maximizing sperm metabolism and performance and, ultimately, the fertilizing capacity of spermatozoa.

  4. Hyaluronidase 2: A Novel Germ Cell Hyaluronidase with Epididymal Expression and Functional Roles in Mammalian Sperm1

    PubMed Central

    Modelski, Mark J.; Menlah, Gladys; Wang, Yipei; Dash, Soma; Wu, Kathie; Galileo, Deni S.; Martin-DeLeon, Patricia A.

    2014-01-01

    ABSTRACT To initiate the crucial cell adhesion events necessary for fertilization, sperm must penetrate extracellular matrix barriers containing hyaluronic acid (HA), a task thought to be accomplished by neutral-active hyaluronidases. Here we report that the ∼57 kDa hyaluronidase 2 (HYAL2) that in somatic tissues has been highly characterized to be acid-active is present in mouse and human sperm, as detected by Western blot, flow cytometric, and immunoprecipitation assays. Immunofluorescence revealed its presence on the plasma membrane over the acrosome, the midpiece, and proximal principal piece in mice where protein fractionation demonstrated a differential distribution in subcellular compartments. It is significantly more abundant in the acrosome-reacted (P = 0.04) and soluble acrosomal fractions (P = 0.006) (microenvironments where acid-active hyaluronidases function) compared to that of the plasma membrane where neutral hyaluronidases mediate cumulus penetration. Using HA substrate gel electrophoresis, immunoprecipitated HYAL 2 was shown to have catalytic activity at pH 4.0. Colocalization and coimmunoprecipitation assays reveal that HYAL2 is associated with its cofactor, CD44, consistent with CD44-dependent HYAL2 activity. HYAL2 is also present throughout the epididymis, where Hyal2 transcripts were detected, and in the epididymal luminal fluids. In vitro assays demonstrated that HYAL2 can be acquired on the sperm membrane from epididymal luminal fluids, suggesting that it plays a role in epididymal maturation. Because similar biphasic kinetics are seen for HYAL2 and SPAM1 (Sperm adhesion molecule 1), it is likely that HYAL2 plays a redundant role in the catalysis of megadalton HA to its 20 kDa intermediate during fertilization. PMID:25232017

  5. Spermatogenesis of the green-lipped mussel Perna viridis with dual patterns of acrosome and tail development in spermatids

    NASA Astrophysics Data System (ADS)

    Reunov, A. A.; Au, D. W. T.; Wu, R. S. S.

    1999-08-01

    Spermatogenesis in the mussel Perna viridis was studied by electron microscopy. Results demonstrated that cytological development in spermatogonia and spermatocytes was similar to that previously described in other Mytilidae. Acrosome formation began with the arising of proacrosomal vesicles in spermatogonia. The abundance of proacrosomal vesicles increased in spermatocytes, which were flagellated. However, during spermiogenesis, dual patterns of acrosome development as well as flagellum development could be found among spermatids in a male gonad. The two lines of acrosome formation in spermatids ultimately gave rise to morphologically similar acrosomes. The two lines of flagellum development in spermatids resulted in the formation of sperm cells with either a typically posteriorly directed tail or an anteriorly directed tail.

  6. Sperm Patch-Clamp

    PubMed Central

    Lishko, Polina; Clapham, David E.; Navarro, Betsy; Kirichok, Yuriy

    2014-01-01

    Sperm intracellular pH and calcium concentration ([Ca2+]i) are two central factors that control sperm activity within the female reproductive tract. As such, the ion channels of the sperm plasma membrane that alter intracellular sperm [Ca2+] and pH play important roles in sperm physiology and the process of fertilization. Indeed, sperm ion channels regulate sperm motility, control sperm chemotaxis toward the egg in some species, and may trigger the acrosome reaction. Until recently, our understanding of these important molecules was rudimentary due to the inability to patch-clamp spermatozoa and directly record the activity of these ion channels under voltage clamp. Recently, we overcame this technical barrier and developed a method for reproducible application of the patch-clamp technique to mouse and human spermatozoa. This chapter covers important aspects of application of the patch-clamp technique to spermatozoa, such as selection of the electrophysiological equipment, isolation of spermatozoa for patch-clamp experiments, formation of the gigaohm seal with spermatozoa, and transition into the whole-cell mode of recording. We also discuss potential pitfalls in application of the patch-clamp technique to flagellar ion channels. PMID:23522465

  7. ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation*

    PubMed Central

    Pelletán, Leonardo E.; Suhaiman, Laila; Vaquer, Cintia C.; Bustos, Matías A.; De Blas, Gerardo A.; Vitale, Nicolas; Mayorga, Luis S.; Belmonte, Silvia A.

    2015-01-01

    Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization. PMID:25713146

  8. Spink13, an Epididymis-specific Gene of the Kazal-type Serine Protease Inhibitor (SPINK) Family, Is Essential for the Acrosomal Integrity and Male Fertility*

    PubMed Central

    Ma, Li; Yu, Heguo; Ni, Zimei; Hu, Shuanggang; Ma, Wubin; Chu, Chen; Liu, Qiang; Zhang, Yonglian

    2013-01-01

    Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives. PMID:23430248

  9. Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle.

    PubMed

    Kishida, Kazumi; Sakase, Mitsuhiro; Minami, Kenta; Arai, Miyuki M; Syoji, Reiko; Kohama, Namiko; Akiyama, Takayuki; Oka, Akio; Harayama, Hiroshi; Fukushima, Moriyuki

    2015-01-01

    The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes.

  10. Effects of acrosomal conditions of frozen-thawed spermatozoa on the results of artificial insemination in Japanese Black cattle

    PubMed Central

    KISHIDA, Kazumi; SAKASE, Mitsuhiro; MINAMI, Kenta; ARAI, Miyuki M.; SYOJI, Reiko; KOHAMA, Namiko; AKIYAMA, Takayuki; OKA, Akio; HARAYAMA, Hiroshi; FUKUSHIMA, Moriyuki

    2015-01-01

    The purposes of this study were to examine the relationship between male artificial insemination (AI) fertility and sperm acrosomal conditions assessed by new and conventional staining techniques and to identify possible reproductive dysfunctions causing low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions in Japanese Black bulls. We investigated individual differences among bulls in the results concerning (1) acrosomal conditions of frozen-thawed spermatozoa as assessed by not merely peanut agglutinin-lectin staining (a conventional staining technique) but also immunostaining of acrosomal tyrosine-phosphorylated proteins (a new staining technique), (2) routine AI using frozen-thawed spermatozoa as assessed by pregnancy diagnosis, (3) in vivo fertilization of frozen-thawed spermatozoa and early development of fertilized eggs as assessed by superovulation/AI-embryo collection tests and (4) in vitro fertilization of frozen-thawed spermatozoa with oocytes. The percentages of frozen-thawed spermatozoa with normal acrosomal conditions assessed by the abovementioned staining techniques were significantly correlated with the conception rates of routine AI, rates of transferable embryos in superovulation/AI-embryo collection tests and in vitro fertilization rates. These results are consistent with new suggestions that the distribution of acrosomal tyrosine-phosphorylated proteins as well as the acrosomal morphology of frozen-thawed spermatozoa are AI fertility-associated markers that are valid for the prediction of AI results and that low conception rates in AI using frozen-thawed spermatozoa with poor acrosomal conditions result from reproductive dysfunctions in the processes between sperm insemination into females and early embryo development, probably failed fertilization of frozen-thawed spermatozoa with oocytes. PMID:26300347

  11. Cryopreservation of epididymal sperm in tree shrews (Tupaia belangeri).

    PubMed

    Ping, S; Wang, F; Zhang, Y; Wu, C; Tang, W; Luo, Y; Yang, S

    2011-07-01

    Cryopreservation of sperm from tree shrews, which are considered primitive primates, would enhance genetic management and breeding programs. Epididymal sperm were surgically harvested from male tree shrews, cryopreserved in two Tes-Tris-based cryodiluents, and used in four experiments. In Experiment 1, there were no significant differences in motility and acrosome integrity among five concentrations of egg yolk in TTE after cooling to 4 °C. However, sperm frozen in TTE containing 20% egg yolk at -172 °C/min had better (P < 0.05) post-thaw motility and acrosome integrity. In Experiment 2, sperm held for 10 min prior to storage in liquid nitrogen had greater motility than those held for 5 or 15 min (P < 0.05), but acrosome integrity was not different (P > 0.05) among treatments. In Experiment 3, sperm frozen in TTE diluent had higher (P < 0.05) motility and acrosome integrity than those in TEST diluent. In Experiment 4, there were no differences (P > 0.05) in the fertilization rate of oocytes and the proportion of tree shrews yielding fertilized oocytes, following AI with fresh versus frozen sperm. In conclusion, tree shrew epididymal sperm were successfully cryopreserved, as assessed by post-thaw motility, acrosome integrity, and fertilizing ability.

  12. Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome

    PubMed Central

    Yoon, Sung-Jae; Rahman, Md Saidur; Kwon, Woo-Sung; Park, Yoo-Jin; Pang, Myung-Geol

    2016-01-01

    Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation. PMID:27031703

  13. Addition of Cryoprotectant Significantly Alters the Epididymal Sperm Proteome.

    PubMed

    Yoon, Sung-Jae; Rahman, Md Saidur; Kwon, Woo-Sung; Park, Yoo-Jin; Pang, Myung-Geol

    2016-01-01

    Although cryopreservation has been developed and optimized over the past decades, it causes various stresses, including cold shock, osmotic stress, and ice crystal formation, thereby reducing fertility. During cryopreservation, addition of cryoprotective agent (CPA) is crucial for protecting spermatozoa from freezing damage. However, the intrinsic toxicity and osmotic stress induced by CPA cause damage to spermatozoa. To identify the effects of CPA addition during cryopreservation, we assessed the motility (%), motion kinematics, capacitation status, and viability of epididymal spermatozoa using computer-assisted sperm analysis and Hoechst 33258/chlortetracycline fluorescence staining. Moreover, the effects of CPA addition were also demonstrated at the proteome level using two-dimensional electrophoresis. Our results demonstrated that CPA addition significantly reduced sperm motility (%), curvilinear velocity, viability (%), and non-capacitated spermatozoa, whereas straightness and acrosome-reacted spermatozoa increased significantly (p < 0.05). Ten proteins were differentially expressed (two decreased and eight increased) (>3 fold, p < 0.05) after CPA, whereas NADH dehydrogenase flavoprotein 2, f-actin-capping protein subunit beta, superoxide dismutase 2, and outer dense fiber protein 2 were associated with several important signaling pathways (p < 0.05). The present study provides a mechanistic basis for specific cryostresses and potential markers of CPA-induced stress. Therefore, these might provide information about the development of safe biomaterials for cryopreservation and basic ground for sperm cryopreservation.

  14. [Updated detection of the function of sperm plasma membrane].

    PubMed

    Zhou, Xin; Xia, Xin-Yi; Huang, Yu-Feng

    2010-08-01

    The sperm plasma membrane is rich in polyunsaturated fatty acids and a variety of proteins, and its function is associated with sperm capacitation, acrosome reaction and sperm-egg fusion. Sperm fertilizability can be predicted by detecting the function of the sperm plasma membrane, which is performed mainly with the following five techniques: sperm hypoosmotic swelling test, Eosin gamma water test, sperm membrane lipid peroxidation determination, seminal plasma superoxide dismutase determination, and flow cytometry. The evaluation of the function of sperm plasma membrane can be applied in detecting semen quality, selecting semen centrifugation, assessing the quality and fertilizability of sex-sorted sperm, improving cryopreservation, and guiding the optimization of intracytoplasmic sperm injection. This review presents an update on the principles, methods and steps of the detection of sperm plasma membrane function, as well as an overview of its status quo and application.

  15. Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa.

    PubMed

    O'Bryan, M K; Sebire, K; Meinhardt, A; Edgar, K; Keah, H H; Hearn, M T; De Kretser, D M

    2001-01-01

    Previously we reported the cloning of a member of the cysteine-rich secretory protein family, tpx-1, from a testis expression library using an outer dense fiber (ODF)-specific antiserum. Using immunohistochemical and immunoelectron microscopic techniques and Western blotting of purified sperm tail components, we have determined that tpx-1 exists as 25 and 27 kDa proteins in two components of rat spermatid: the ODFs and the acrosome. Tpx-1 mRNA is first expressed in the late pachytene spermatocytes, but the production of these tpx-1 proteins is translationally delayed for 4-5 days before being incorporated into the developing sperm acrosome, surrounding the elongating and condensing spermatid nucleus. Concurrent with sperm head formation, tpx-1 protein was incorporated into the developing sperm tail, and specifically the ODFs. The tpx-1 protein was seen within structures resembling granulated bodies in the cytoplasmic lobe of elongating spermatids and was incorporated subsequently into the growing tail in a manner consistent with ODF development. In addition, tpx-1 protein was localized at the ultrastructural level of the connecting piece of the neck and longitudinal columns of the fibrous sheath, suggesting common protein components in these cytoskeletal structures. As such, tpx-1 may have functional significance in the processes of sperm head development and tail function.

  16. Intracellular pH in sperm physiology.

    PubMed

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L; Darszon, Alberto

    2014-08-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca(2+) channel; Slo3, a K(+) channel; the sperm-specific Na(+)/H(+) exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction.

  17. Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model.

    PubMed

    Simón, Layla; Funes, Abi K; Yapur, Martín A; Cabrillana, María E; Monclus, María A; Boarelli, Paola V; Vincenti, Amanda E; Saez Lancellotti, Tania E; Fornés, Miguel W

    2017-01-01

    Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis-the last step of spermatogenesis involved in sperm shaping-was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin-alpha-tubulin-GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that hypercholesterolemic

  18. Manchette-acrosome disorders during spermiogenesis and low efficiency of seminiferous tubules in hypercholesterolemic rabbit model

    PubMed Central

    Simón, Layla; Funes, Abi K.; Yapur, Martín A.; Cabrillana, María E.; Monclus, María A.; Boarelli, Paola V.; Vincenti, Amanda E.

    2017-01-01

    Hypercholesterolemia is a marker for several adult chronic diseases. Recently we demonstrated that sub/infertility is also associated to Hypercholesterolemia in rabbits. Seminal alterations included: abnormal sperm morphology, decreased sperm number and declined percentage of motile sperm, among others. In this work, our objective was to evaluate the effects of hypercholesterolemia on testicular efficiency and spermiogenesis, as the latter are directly related to sperm number and morphology respectively. Tubular efficiency was determined by comparing total number of spermatogenic cells with each cell type within the proliferation/differentiation compartments. We found lower testicular efficiency related to both a decrease in spermatogonial cells and an increase in germ cell apoptosis in hypercholesterolemic rabbits. On the other hand, spermiogenesis–the last step of spermatogenesis involved in sperm shaping–was detaily analyzed, particularly the acrosome-nucleus-manchette complex. The manchette is a microtubular-based temporary structure responsible in sperm cell elongation. We analyzed the contribution of actin filaments and raft microdomains in the arrangement of the manchette. Under fluorescence microscopy, spermatocyte to sperm cell development was followed in cells isolated from V to VIII tubular stages. In cells from hypercholesterolemic rabbits, abnormal development of acrosome, nucleus and inaccurate tail implantation were associated with actin–alpha-tubulin–GM1 sphingolipid altered distribution. Morphological alterations were also observed at electron microscopy. We demonstrated for the first time that GM1-enriched microdomains together with actin filaments and microtubules are involved in allowing the correct anchoring of the manchette complex. In conclusion, cholesterol enriched diets promote male fertility alterations by affecting critical steps in sperm development: spermatogenesis and spermiogenesis. It was also demonstrated that

  19. Albumin Is Synthesized in Epididymis and Aggregates in a High Molecular Mass Glycoprotein Complex Involved in Sperm-Egg Fertilization

    PubMed Central

    Souza, Gustavo Henrique Martins Ferreira; Tanaka, Hiromitsu; Eberlin, Marcos Nogueira; Hyslop, Stephen; Alvares, Lúcia Elvira; Pereira, Luís Antonio Violin Dias

    2014-01-01

    The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization. PMID:25084016

  20. Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization.

    PubMed

    Arroteia, Kélen Fabíola; Barbieri, Mainara Ferreira; Souza, Gustavo Henrique Martins Ferreira; Tanaka, Hiromitsu; Eberlin, Marcos Nogueira; Hyslop, Stephen; Alvares, Lúcia Elvira; Pereira, Luís Antonio Violin Dias

    2014-01-01

    The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb) TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa) that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

  1. Ca2+-stores in sperm: their identities and functions

    PubMed Central

    Costello, Sarah; Michelangeli, Francesco; Nash, Kate; Lefievre, Linda; Morris, Jennifer; Machado-Oliveira, Gisela; Barratt, Christopher; Kirkman-Brown, Jackson; Publicover, Stephen

    2013-01-01

    Intracellular Ca2+ stores play a central role in the regulation of cellular [Ca2+]i and the generation of complex [Ca2+] signals such as oscillations and waves. Ca2+ signalling is of particular significance in sperm cells, where it is a central regulator in many key activities (including capacitation, hyperactivation, chemotaxis and acrosome reaction) yet mature sperm lack endoplasmic reticulum and several other organelles which serve as Ca2+ stores in somatic cells. Here we review (i) the evidence for the expression in sperm of the molecular components (pumps and channels) which are functionally significant in the activity of Ca2+ stores of somatic cells and (ii) the evidence for the existence of functional Ca2+ stores in sperm. This evidence supports the existence of at least two storage organelles in mammalian sperm, one in the acrosomal region and another in the region of the sperm neck and midpiece. We then go on to discuss the likely identity of these organelles and their discrete functions: regulation by the acrosome of its own secretion and regulation by membranous organelles at the sperm neck (and possibly by the mitochondria) of flagellar activity and hyperactivation. Finally we consider the ability of the sperm discretely to control mobilisation of these stores and the functional interaction of stored Ca2+ at the sperm neck/midpiece with CatSper channels in the principal piece in regulation of the activities of mammalian sperm. PMID:19542252

  2. Sperm function test

    PubMed Central

    Talwar, Pankaj; Hayatnagarkar, Suryakant

    2015-01-01

    With absolute normal semen analysis parameters it may not be necessary to shift to specialized tests early but in cases with borderline parameters or with history of fertilization failure in past it becomes necessary to do a battery of tests to evaluate different parameters of spermatozoa. Various sperm function tests are proposed and endorsed by different researchers in addition to the routine evaluation of fertility. These tests detect function of a certain part of spermatozoon and give insight on the events in fertilization of the oocyte. The sperms need to get nutrition from the seminal plasma in the form of fructose and citrate (this can be assessed by fructose qualitative and quantitative estimation, citrate estimation). They should be protected from the bad effects of pus cells and reactive oxygen species (ROS) (leukocyte detection test, ROS estimation). Their number should be in sufficient in terms of (count), structure normal to be able to fertilize eggs (semen morphology). Sperms should have intact and functioning membrane to survive harsh environment of vagina and uterine fluids (vitality and hypo-osmotic swelling test), should have good mitochondrial function to be able to provide energy (mitochondrial activity index test). They should also have satisfactory acrosome function to be able to burrow a hole in zona pellucida (acrosome intactness test, zona penetration test). Finally, they should have properly packed DNA in the nucleus to be able to transfer the male genes (nuclear chromatic decondensation test) to the oocyte during fertilization. PMID:26157295

  3. Molecular architecture of the human sperm IZUMO1 and egg JUNO fertilization complex.

    PubMed

    Aydin, Halil; Sultana, Azmiri; Li, Sheng; Thavalingam, Annoj; Lee, Jeffrey E

    2016-06-23

    Fertilization is an essential biological process in sexual reproduction and comprises a series of molecular interactions between the sperm and egg. The fusion of the haploid spermatozoon and oocyte is the culminating event in mammalian fertilization, enabling the creation of a new, genetically distinct diploid organism. The merger of two gametes is achieved through a two-step mechanism in which the sperm protein IZUMO1 on the equatorial segment of the acrosome-reacted sperm recognizes its receptor, JUNO, on the egg surface. This recognition is followed by the fusion of the two plasma membranes. IZUMO1 and JUNO proteins are indispensable for fertilization, as constitutive knockdown of either protein results in mice that are healthy but infertile. Despite their central importance in reproductive medicine, the molecular architectures of these proteins and the details of their functional roles in fertilization are not known. Here we present the crystal structures of human IZUMO1 and JUNO in unbound and bound conformations. The human IZUMO1 structure exhibits a distinct boomerang shape and provides structural insights into the IZUMO family of proteins. Human IZUMO1 forms a high-affinity complex with JUNO and undergoes a major conformational change within its N-terminal domain upon binding to the egg-surface receptor. Our results provide insights into the molecular basis of sperm-egg recognition, cross-species fertilization, and the barrier to polyspermy, thereby promising benefits for the rational development of non-hormonal contraceptives and fertility treatments for humans and other mammals.

  4. Role of Wasp and the small GTPases RhoA, RhoB, and Cdc42 during capacitation and acrosome reaction in spermatozoa of English guinea pigs.

    PubMed

    Delgado-Buenrostro, Norma L; Mújica, Adela; Chiquete-Felix, Natalia; Déciga-Alcaraz, Alejandro; Medina-Reyes, Estefany I; Uribe-Carvajal, Salvador; Chirino, Yolanda I

    2016-10-01

    Cytoskeleton remodeling is necessary for capacitation and the acrosome reaction in spermatozoa. F-actin is located in the acrosome and equatorial region during capacitation, but is relocated in the post-acrosomal region during the acrosome reaction in spermatozoa from bull, rat, mice, and guinea pig. Actin polymerization and relocalization are generally regulated by small GTPases that activate Wasp protein, which coordinates with Arp2/3, profilin I, and profilin II to complete cytoskeletal remodeling. This sequence of events is not completely described in spermatozoa, though. Therefore, the aim of this study was to determine if Wasp interacts with small GTPases (RhoA, RhoB, and Cdc42) and proteins (Arp2/3, profilin I, and profilin II) that co-localize with F-actin during capacitation and the acrosome reaction in English guinea pig spermatozoa obtained from the vas deferens. The spermatozoa were capacitated in calcium-free medium, incubated with an activator or an inhibitor of GTPases, and then induced to acrosome react using calcium. The distribution patterns of F-actin were compared to the patterns of Wasp and its putative interaction partners: Wasp and RhoB, but not RhoA or Cdc42, localization overlap with F-actin during capacitation and the acrosome reaction. Activation of small GTPases localized RhoB to the post-acrosomal region whereas their inhibition prevented acrosome exocytosis. Arp2/3 and profilin II appear to interact with Wasp in the post-acrosomal region and flagellum, while profilin I and Wasp could be found in the equatorial region. Thus, Wasp and F-actin distribution overlap during capacitation and acrosome reaction, and small GTPases play an important role in cytoskeleton remodeling during these processes in spermatozoa. Mol. Reprod. Dev. 83: 927-937, 2016 © 2016 Wiley Periodicals, Inc.

  5. Cysteamine supplementation revealed detrimental effect on cryosurvival of buffalo sperm based on computer-assisted semen analysis and oxidative parameters.

    PubMed

    Swami, Dheer Singh; Kumar, Pradeep; Malik, R K; Saini, Monika; Kumar, Dharmendra; Jan, M H

    2017-02-01

    The aim of this study was to investigate the effect of addition of cysteamine to the semen extender on post-thaw semen quality. A total of 30 ejaculates were collected from six bulls. Each ejaculate was divided into five equal parts and diluted to final concentration of 80 million sperms/mL using Optixcell(®)(IMV, France) semen extender supplemented with different concentrations of cysteamine (0, 0.75, 1.25, 2.5 and 5mM) and cryopreserved. In the frozen-thawed samples, the VAP, VSL, VCL ALH and sperm motility of control samples was greater (P<0.05) than cysteamine treated samples. The sperm abnormality and malondialdehyde (MDA) concentration were found highest in 5mM cysteamine treated samples. The cysteamine treated samples travelled significantly less distance in cervical mucus as compared to control. Further, cysteamine decreased acrosomal integrity of sperm. In incubation test, control samples showed better sperm motility as compared to treatment groups. Further, cysteamine supplementation decreased the total antioxidants and increased the MDA concentration of sperm. From the study, we hypothesized that cysteamine cannot stimulate synthesis of glutathione (GSH) intracellularly in sperm to combat free radicals because during the maturation, sperm lost its cytoplasm which is necessary for biochemical reaction in which cysteamine reacts with cystine to form a mixed disulfide which taken up by cells and split into cysteine in the cytoplasm. Synthesis of GSH depends on the availability of cysteine. In conclusion, the results of our study strongly emphasize that cysteamine would not be a suitable additive in extender for freezing buffalo bull semen.

  6. Sperm morphology: consistency of assessment of the same sperm by different observers.

    PubMed

    Baker, H W; Clarke, G N

    1987-01-01

    Photographic slides of 36 sperm were shown to a group of 28 observers with different lengths of experience in assessing sperm morphology. Experienced observers were generally consistent (10 or more of the 17 agreeing) in classifying sperm as normal, amorphous, small heads, lacking acrosomes, and having tail defects or cytoplasmic droplets but categorization was more variable for large, tapering and pyriform heads. This study highlights the need for more widespread agreement about definition of sperm shapes and the development of practical objective methods of assessment.

  7. Dynamics of Sun5 Localization during Spermatogenesis in Wild Type and Dpy19l2 Knock-Out Mice Indicates That Sun5 Is Not Involved in Acrosome Attachment to the Nuclear Envelope

    PubMed Central

    Yassine, Sandra; Escoffier, Jessica; Nahed, Roland Abi; Pierre, Virginie; Karaouzene, Thomas; Ray, Pierre F.; Arnoult, Christophe

    2015-01-01

    The acrosome is an organelle that is central to sperm physiology and a defective acrosome biogenesis leads to globozoospermia, a severe male infertility. The identification of the actors involved in acrosome biogenesis is therefore particularly important to decipher the molecular pathogeny of globozoospermia. We recently showed that a defect in the DPY19L2 gene is present in more than 70% of globozoospermic men and demonstrated that Dpy19l2, located in the inner nuclear membrane, is the first protein involved in the attachment of the acrosome to the nuclear envelope (NE). SUN proteins serve to link the nuclear envelope to the cytoskeleton and are therefore good candidates to participate in acrosome-nucleus attachment, potentially by interacting with DPY19L2. In order to characterize new actors of acrosomal attachment, we focused on Sun5 (also called Spag4l), which is highly expressed in male germ cells, and investigated its localization during spermatogenesis. Using immunohistochemistry and Western blot experiments in mice, we showed that Sun5 transits through different cellular compartments during meiosis. In pachytene spermatocytes, it is located in a membranous compartment different to the reticulum. In round spermatids, it progresses to the Golgi and the NE before to be located to the tail/head junction in epididymal sperm. Interestingly, we demonstrate that Sun5 is not, as initially reported, facing the acrosome but is in fact excluded from this zone. Moreover, we show that in Dpy19l2 KO spermatids, upon the detachment of the acrosome, Sun5 relocalizes to the totality of the NE suggesting that the acrosome attachment excludes Sun5 from the NE facing the acrosome. Finally, Western-blot experiments demonstrate that Sun5 is glycosylated. Overall, our work, associated with other publications, strongly suggests that the attachment of the acrosome to the nucleus does not likely depend on the formation of SUN complexes. PMID:25775128

  8. SpermBlue: a new universal stain for human and animal sperm which is also amenable to automated sperm morphology analysis.

    PubMed

    van der Horst, G; Maree, L

    2009-12-01

    Our study was aimed at exploring a simple procedure to stain differentially the acrosome, head, midpiece, and flagellum of human and animal sperm. A further prerequisite was that sperm morphology of the stained samples could be analyzed using automated sperm morphology analysis (ASMA). We developed a new staining process using SpermBlue fixative and SpermBlue stain, which are iso-osmotic in relation to semen. The entire fixation and staining processes requires only 25 min. Three main steps are required. First, a routine sperm smear is made by either using semen or sperm in a diluting medium. The smear is allowed to air dry at room temperature. Second, the smear is fixed for 10 min by either placing the slide with the dried smear in a staining tray containing SpermBlue fixative or by adding 1 ml SpermBlue fixative to the slide. Third, the fixed smear is stained for 15 min by either immersing the slide in a staining tray containing SpermBlue stain or adding four drops of SpermBlue stain to the fixed smear. The stained slide is dipped gently in distilled water followed by air drying and mounting in DPX or an equivalent medium. The method is simple and suitable for field conditions. Sperm of human, three monkey species, horse, boar, bull, ram, mouse, rat, domestic chicken, fish, and invertebrate species were stained successfully using the SpermBlue staining process. SpermBlue stains human and animal sperm different hues or intensities of blue. It is possible to distinguish clearly the acrosome, sperm head, midpiece, principal piece of the tail, and even the short end piece. The Sperm Class Analyzer ASMA system was used successfully to quantify sperm head and midpiece measurements automatically at either 600 x or 1000 x magnification for most of the species studied.

  9. Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization

    PubMed Central

    Lee, Won Young; Lee, Ran; Kim, Hee Chan; Lee, Kyung Hoon; Cui, Xiang Shun; Kim, Nam Hyung; Kim, Sang Hyun; Lee, Il Joo; Uhm, Sang Jun; Yoon, Min Jung; Song, Hyuk

    2014-01-01

    The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not

  10. Pig Spermatozoa Defect in Acrosome Formation Caused Poor Motion Parameters and Fertilization Failure through Artificial Insemination and In vitro Fertilization.

    PubMed

    Lee, Won Young; Lee, Ran; Kim, Hee Chan; Lee, Kyung Hoon; Cui, Xiang Shun; Kim, Nam Hyung; Kim, Sang Hyun; Lee, Il Joo; Uhm, Sang Jun; Yoon, Min Jung; Song, Hyuk

    2014-10-01

    The selection of morphologically normal spermatozoa is critical to obtain high breeding performances in boar breeding farms and artificial insemination (AI) centers. Parameters for the selection of semen mainly include total sperm motility, concentration, and morphology. However, these primary parameters are often not reliable for discriminating between normal and abnormal, non-fertilizable spermatozoa. The present study was designed to compare the motion characteristics, fertilization ability using in vitro fertilization (IVF), and acrosome formation of the semen from boars having low (boar number 2012) and normal (boar number 2004 and 2023) breeding performances. The ultimate goal was to identify additional simple and easy criteria for the selection of normal sperm. There was no significant difference between boar 2004 and boar 2023 sperm total motility in computer assisted sperm analysis. However, boar number 2012 semen presented a significantly reduced population of rapid moving spermatozoa and an increased population of slow moving spermatozoa compared to boar numbers 2004 and 2023. Analysis of detailed motion characteristics revealed that sperm from boar number 2012 had significantly reduced motility in progressiveness, average path velocity, straight-line velocity (VSL), curvilinear velocity (VCL), straightness, and linearity. The assessment of the fertilizing ability by IVF also showed that sperm from boar number 2012 showed a fertility rate of 3.4%, whereas sperm from boar number 2023 had a fertility rate of 75.45%. Interestingly, most of the sperm nuclei were found on the peripheral area of the oocytes, suggesting that the sperm from boar number 2012 lacked penetration ability into the oocyte zonapellucida. The acrosome formation analysis using Pisum sativum agglutinin staining demonstrated that the sperm from boar number 2012 had a defect in acrosome formation. Consequently, primary parameters for selecting semen before AI such as motility are not

  11. Platelet-activating factor in Iberian pig spermatozoa: receptor expression and role as enhancer of the calcium-induced acrosome reaction.

    PubMed

    Bragado, M J; Gil, M C; Garcia-Marin, L J

    2011-12-01

    Platelet-activating factor (PAF) is a phospholipid involved in reproductive physiology. PAF receptor is expressed in some mammalian spermatozoa species where it plays a role in these germ-cell-specific processes. The aim of this study is to identify PAF receptor in Iberian pig spermatozoa and to evaluate PAF's effects on motility, viability and acrosome reaction. Semen samples from Iberian boars were used. PAF receptor identification was performed by Western blotting. Spermatozoa motility was analysed by computer-assisted sperm analysis system, whereas spermatozoa viability and acrosome reaction were evaluated by flow cytometry. Different PAF concentrations added to non-capacitating medium during 60 min have no effect on any spermatozoa motility parameter measured. Acrosome reaction was rapid and potently induced by 1 μm calcium ionophore A23187 showing an effect at 60 min and maximum at 240 min. PAF added to a capacitating medium is not able to induce spermatozoa acrosome reaction at any time studied. However, PAF, in the presence of A23187, significantly accelerates and enhances the calcium-induced acrosome reaction in a concentration-dependent manner in Iberian boar spermatozoa. Exogenous PAF does not affect at all spermatozoa viability, whereas slightly exacerbated the A23187-induced loss in viability. This work demonstrates that PAF receptor is expressed in Iberian pig spermatozoa and that its stimulation by PAF regulates the calcium-induced acrosome reaction. This work contributes to further elucidate the physiological regulation of the most relevant spermatozoa functions for successful fertilization: acrosome reaction.

  12. Characterization of a novel human sperm-associated antigen 9 (SPAG9) having structural homology with c-Jun N-terminal kinase-interacting protein

    PubMed Central

    Jagadish, Nirmala; Rana, Ritu; Selvi, Ramasamy; Mishra, Deepshikha; Garg, Manoj; Yadav, Shikha; Herr, John C.; Okumura, Katsuzumi; Hasegawa, Akiko; Koyama, Koji; Suri, Anil

    2005-01-01

    We report a novel SPAG9 (sperm-associated antigen 9) protein having structural homology with JNK (c-Jun N-terminal kinase)-interacting protein 3. SPAG9, a single copy gene mapped to the human chromosome 17q21.33 syntenic with location of mouse chromosome 11, was earlier shown to be expressed exclusively in testis [Shankar, Mohapatra and Suri (1998) Biochem. Biophys. Res. Commun. 243, 561–565]. The SPAG9 amino acid sequence analysis revealed identity with the JNK-binding domain and predicted coiled-coil, leucine zipper and transmembrane domains. The secondary structure analysis predicted an α-helical structure for SPAG9 that was confirmed by CD spectra. Microsequencing of higher-order aggregates of recombinant SPAG9 by tandem MS confirmed the amino acid sequence and mono atomic mass of 83.9 kDa. Transient expression of SPAG9 and its deletion mutants revealed that both leucine zipper with extended coiled-coil domains and transmembrane domain of SPAG9 were essential for dimerization and proper localization. Studies of MAPK (mitogenactivated protein kinase) interactions demonstrated that SPAG9 interacted with higher binding affinity to JNK3 and JNK2 compared with JNK1. No interaction was observed with p38α or extracellular-signal-regulated kinase pathways. Polyclonal antibodies raised against recombinant SPAG9 recognized native protein in human sperm extracts and localized specifically on the acrosomal compartment of intact human spermatozoa. Acrosome-reacted spermatozoa demonstrated SPAG9 immunofluorescence, indicating its retention on the equatorial segment after the acrosome reaction. Further, anti-SPAG9 antibodies inhibited the binding of human spermatozoa to intact human oocytes as well as to matched hemizona. This is the first report of sperm-associated JNK-binding protein that may have a role in spermatozoa–egg interaction. PMID:15693750

  13. Autometallographic demonstration of zinc ions in rat sperm cells.

    PubMed

    Stoltenberg, M; Sørensen, M B; Danscher, G; Juhl, S; Andreasen, A; Ernst, E

    1997-09-01

    An in-vitro technique for autometallographic (AMG) demonstration of chelatable zinc in electroejaculated sperm cells and spermatozoa from the epididymis is presented and the localization of zinc ions in rat spermatozoa is described. Sperm cells from caput epididymis showed zinc staining in all parts of the tail and a sparse, dispersed staining in the acrosome. Spermatozoa from cauda epididymis showed heavy staining in the acrosome but no staining in the tail, or post-acrosomal part of the sperm head. This distinct acrosomal AMG staining was also found in ejaculated spermatozoa, but additionally a segmentation of the tail was seen based on differences in staining intensity. The membrane penetrating chelator diethyldithiocarbamate (DEDTC) was found to block the AMG staining whereas calcium-EDTA, known not to pass through cell membranes, did not influence the staining, proving that the detected zinc ions are intracellularly located. Two different approaches for demonstrating the presence of a chelatable zinc pool at electron microscope levels are presented, and the ultrastructural presence of AMG grains located in the acrosome and in the mitochondria of the midpiece is demonstrated. It is postulated that an exchange of zinc ions takes place between the epididymal epithelium and the sperm cells as they pass along the epididymal duct.

  14. Trehalose enhances osmotic tolerance and suppresses lysophosphatidylcholine-induced acrosome reaction in ram spermatozoon.

    PubMed

    Ahmad, E; Naseer, Z; Aksoy, M; Küçük, N; Uçan, U; Serin, I; Ceylan, A

    2015-09-01

    This study was aimed to investigate the influence of trehalose on osmotic tolerance and the ability of ram spermatozoon to undergo acrosome reaction induced by lysophosphatidylcholine (LPC). In experiment 1, the diluted ejaculates were exposed to anisosmotic fructose solutions (70, 500, 750 and 1000 mOsm l(-1) ) with or without 50 mm trehalose. The presence of trehalose in hyperosmotic conditions enhanced (P < 0.05) the percentage of live, live-intact and intact spermatozoa. Similarly, trehalose enhanced (P < 0.05) the live and live-intact spermatozoa during hypo-osmotic conditions. In experiment 2, the centrifuged ejaculates were diluted with TCG only or TCG containing either 50 or 100 mm trehalose. The acrosome reaction was induced by LPC. The percentage of acrosome-reacted spermatozoon was less (P < 0.05) in trehalose-supplemented groups compared to control. In experiment 3, the ejaculates were cryopreserved in an extender containing 0 mm (control), 50 mm or 100 mm trehalose. Supplementation of extender with trehalose, either 50 mm or 100 mm, enhanced the cryosurvival rate (P < 0.05) compared to the control. In conclusion, the presence of trehalose in anisosmotic conditions enhances the osmotic tolerance, cryosurvival rate of ram spermatozoon and suppresses their ability to undergo LPC and cryo-induced acrosome reaction.

  15. Functional distribution of synapsin I in human sperm

    PubMed Central

    Coleman, William L.; Kulp, Adam C.; Venditti, Jennifer J.

    2015-01-01

    Proteins known to function during cell–cell communication and exocytosis in neurons and other secretory cells have recently been reported in human sperm. Synapsins are a group of proteins that have been very well characterized in neurons, but little is known about synapsin function in other cell types. Based upon previous findings and the known function of synapsin, we tested the hypothesis that synapsin I was present in human sperm. Washed, capacitated, and acrosome induced sperm preparations were used to evaluate the functional distribution of synapsin I using immunocytochemistry. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were used for protein blotting techniques. Immunolocalization revealed synapsin I was enriched in the sperm equatorial segment. Protein extracts from mouse brain, mouse testis/epididymis, and human semen were positive for synapsin I using several different antibodies, and dot blot results were confirmed by Western blot analyses. Finally, treatment of capacitated and acrosome reaction induced samples with anti-synapsin antibodies significantly reduced sperm motility. Localization of synapsin I in human sperm is a novel finding. The association of synapsin I with the sperm equatorial segment and effects on motility are suggestive of a role associated with capacitation and/or acrosome reaction, processes that render sperm capable of fertilizing an oocyte. PMID:26566474

  16. Effect of washing on motility and acrosome morphology of frozen-thawed goat spermatozoa.

    PubMed

    Memon, M A; Bretzlaff, K N; Ott, R S

    1985-02-01

    Semen from 4 bucks was collected using an artificial vagina and was pooled and divided into 6 aliquots. Three aliquots were washed twice, 15 minutes each time, with Ringer's solution, and the fluid was removed by centrifugation at 950 X g between washes. All 6 aliquots (3 washed and 3 unwashed) were extended with skim milk-glycerol, lactose-egg yolk-glycerol, or tris (hydroxymethyl) aminomethane-citric acid-egg yolk-glycerol and were frozen in straws to -196 C. The semen was then thawed and kept at 37 C for 8 hours. Percentage of sperm motility was estimated, and the percentage of normal acrosomes (NA) was determined at 0, 2, 4, 6, and 8 hours after thawing. The experiment was repeated 7 times. The data indicated a significant positive effect (P = 0.0009) of washing on motility, but no effect (P = 0.5347) of extender. There was also a significantly higher percentage of NA in washed semen (P less than 0.0001). Sperm extended in tris aminomethane-citric acid-egg yolk-glycerol had more NA than those extended in lactose-egg yolk-glycerol. Sperm motility and acrosome morphology were depressed also in the presence of seminal plasma for the milk extender, which did not contain egg yolk. Removal of seminal plasma from goat semen was beneficial in preserving the integrity of the spermatozoa after freezing, regardless of the extender used.

  17. Novel Flow Cytometry Analyses of Boar Sperm Viability: Can the Addition of Whole Sperm-Rich Fraction Seminal Plasma to Frozen-Thawed Boar Sperm Affect It?

    PubMed Central

    Díaz, Rommy; Boguen, Rodrigo; Martins, Simone Maria Massami Kitamura; Ravagnani, Gisele Mouro; Leal, Diego Feitosa; Oliveira, Melissa de Lima; Muro, Bruno Bracco Donatelli; Parra, Beatriz Martins; Meirelles, Flávio Vieira; Papa, Frederico Ozanan; Dell’Aqua, José Antônio; Alvarenga, Marco Antônio; Moretti, Aníbal de Sant’Anna; Sepúlveda, Néstor

    2016-01-01

    Boar semen cryopreservation remains a challenge due to the extension of cold shock damage. Thus, many alternatives have emerged to improve the quality of frozen-thawed boar sperm. Although the use of seminal plasma arising from boar sperm-rich fraction (SP-SRF) has shown good efficacy; however, the majority of actual sperm evaluation techniques include a single or dual sperm parameter analysis, which overrates the real sperm viability. Within this context, this work was performed to introduce a sperm flow cytometry fourfold stain technique for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential. We then used the sperm flow cytometry fourfold stain technique to study the effect of SP-SRF on frozen-thawed boar sperm and further evaluated the effect of this treatment on sperm movement, tyrosine phosphorylation and fertility rate (FR). The sperm fourfold stain technique is accurate (R2 = 0.9356, p > 0.01) for simultaneous evaluation of plasma and acrosomal membrane integrity and mitochondrial membrane potential (IPIAH cells). Centrifugation pre-cryopreservation was not deleterious (p > 0.05) for any analyzed variables. Addition of SP-SRF after cryopreservation was able to improve total and progressive motility (p < 0.05) when boar semen was cryopreserved without SP-SRF; however, it was not able to decrease tyrosine phosphorylation (p > 0.05) or improve IPIAH cells (p > 0.05). FR was not (p > 0.05) statistically increased by the addition of seminal plasma, though females inseminated with frozen-thawed boar semen plus SP-SRF did perform better than those inseminated with sperm lacking seminal plasma. Thus, we conclude that sperm fourfold stain can be used to simultaneously evaluate plasma and acrosomal membrane integrity and mitochondrial membrane potential, and the addition of SP-SRF at thawed boar semen cryopreserved in absence of SP-SRF improve its total and progressive motility. PMID:27529819

  18. Effect of sperm pretreatment with sodium hydroxide and dithiothreitol on the efficiency of bovine intracytoplasmic sperm injection.

    PubMed

    Arias, M E; Sánchez, R; Risopatrón, J; Pérez, L; Felmer, R

    2014-01-01

    The efficiency of intracytoplasmic sperm injection (ICSI) in bovines is lower than in other species due, in part, to a lack of optimal conditions for its implementation; this has hindered the achievement of high rates of embryonic development and the birth of live offspring. The aim of the present study was to evaluate the effects of pretreatment of bovine spermatozoa with NaOH and dithiothreitol (DTT) on the viability, plasma membrane integrity, DNA fragmentation and in vitro developmental potential of embryos generated by ICSI. Following pretreatment of spermatozoa with 5 mM DTT for 20 min and a low concentration of NaOH (1 mM for 60 min), there were fewer live and acrosome reacted spermatozoa (44% and 34%, respectively) than in the control group without treatment (82%). Spermatozoa subjected to higher alkali concentrations (10-50 mM) were mostly dead and reacted. However, pronuclear formation, cleavage, blastocyst rate and embryo quality did not differ between these pretreatment groups and the untreated control group. In conclusion, we have described, for the first time, the effects of NaOH treatment on bovine spermatozoa and subsequent in vitro embryonic development after ICSI, and have demonstrated that pretreatment of bovine spermatozoa with NaOH or DTT is not necessary for an appropriate in vitro embryo development in this species.

  19. Protective effect of Rhodiola rosea polysaccharides on cryopreserved boar sperm.

    PubMed

    Yang, Shen-Min; Wang, Ting; Wen, Duan-Gai; Hou, Jian-Quan; Li, Hai-Bo

    2016-01-01

    Cryopreservation brings sublethal damage to sperm, resulting in reduced fertile life of sperm. Rhodiola rosea polysaccharides (RPs) have antiviral, antioxidant and antitumor activities. In the present study, the cryoprotective effect of RPs on boar sperm quality parameters after frozen-thawed process was investigated. Boar sperm was cryopreserved in the extender with RPs added at concentrations of 0 (used as control), 2, 4, 6, 8 and 10mg/L and their effects on the quality of frozen-thawed boar sperm were assessed. Addition of RPs significantly improved sperm motility, mitochondrial activity, acrosomal integrity, plasma membrane integrity, superoxide dismutase and glutathione peroxidase activity and decreased sperm malonaldehyde level (p<0.05). The results indicated that the addition of RPs to the freezing extender decreased the cryodamage to the boar sperm.

  20. Cryotolerance of Sperm from Transgenic Rhesus Macaques (Macaca mulatta)

    PubMed Central

    Moran, Sean P; Chi, Tim; Prucha, Melinda S; Agca, Yuksel; Chan, Anthony WS

    2016-01-01

    Cryopreservation is an important tool routinely used in preserving sperm for assisted reproductive technologies and for genetic preservation of unique animal models. Here we investigated the viability of fresh and frozen sperm from rhesus macaques on the basis of motility, membrane integrity, and acrosome integrity. Sperm motility was determined by visual evaluation; membrane and acrosome integrity were assessed simultaneously through triple staining with Hoechst 33342, propidium iodide, and fluorescein isothiocyanate–peanut agglutinin. We compared thawed semen that had been cryopreserved by using 2 different media with fresh semen from wildtype (WT) macaques; fresh semen from a model of Huntington disease (HD) with fresh WT semen; and fresh HD with cryopreserved-thawed HD semen. Our new freezing media (TEST EQ) preserved the acrosome better, with less net damage, than did traditional TEST (egg yolk extender containing TES and Tris) media. In addition, the percentage of membrane-damaged cells was similar in fresh HD semen (38.6% ± 2.9%) and WT semen (35.5% ±1.9%). Membrane and acrosomal damage were not different between HD and WT sperm after cryopreservation and subsequent thawing. Furthermore, cryopreservation had similar negative effects on the motility of HD and WT sperm. These data illustrate that semen from a rhesus macaque model of HD is similarly cryotoleratant to that from WT animals. PMID:27657705

  1. Labelling of living mammalian spermatozoa with the fluorescent thiol alkylating agent, monobromobimane (MB): immobilization upon exposure to ultraviolet light and analysis of acrosomal status

    SciTech Connect

    Cummins, J.M.; Fleming, A.D.; Crozet, N.; Kuehl, T.J.; Kosower, N.S.; Yanagimachi, R.

    1986-03-01

    Living spermatozoa of seven mammalian species were treated with the thiol-alkylating fluorescent labelling compound, monobromobimane (MBBR). MB-labelling alone had no effect on sperm motility, nor on the time course or ability of golden hamster spermatozoa to undergo the acrosome reaction when capacitated in vitro. Exposure of MB-labelled spermatozoa to ultraviolet (UV) light and excitation of the MB fluorochrome resulted in virtually immediate immobilization of the spermatozoa without affecting acrosomal status. UV exposure of unlabelled spermatozoa for up to 30 sec had no effect upon motility. Immobilization of MB-labelled spermatozoa depended on the midpiece being irradiated, as irradiation of the head alone, or of the more distal parts of the principal piece, had little or no effect upon motility. Labelling with MB followed by immobilization of individually selected spermatozoa was most useful for detailing the course and site of occurrence of the acrosome reaction during penetration of the cumulus oophorus by golden hamster spermatozoa in vitro. In these often hyperactivated spermatozoa, precise determination of the acrosomal status could not often otherwise be made due to the difficulty in visualizing the acrosomal region of a vigorously thrashing, hyperactivated spermatozoon. This technique should prove valuable in a variety of studies on sperm motility, capacitation and fertilization, and could also be extended to other cell systems.

  2. Sphingosine 1-Phosphate and Sphingosine Kinase Are Involved in a Novel Signaling Pathway Leading to Acrosomal Exocytosis*

    PubMed Central

    Suhaiman, Laila; De Blas, Gerardo A.; Obeid, Lina M.; Darszon, Alberto; Mayorga, Luis S.; Belmonte, Silvia A.

    2010-01-01

    Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. Here we report that this lipid triggers acrosomal exocytosis in human sperm by a mechanism involving a Gi-coupled receptor. Real-time imaging showed a remarkable increase of cytosolic calcium upon activation with sphingosine 1-phosphate and pharmacological experiments indicate that the process requires extracellular calcium influx through voltage and store-operated calcium channels and efflux from intracellular stores through inositol 1,4,5-trisphosphate-sensitive calcium channels. Sphingosine 1-phosphate-induced exocytosis requires phospholipase C and protein kinase C activation. We investigated possible sources of the lipid. Western blot indicates that sphingosine kinase 1 is present in spermatozoa. Indirect immunofluorescence showed that phorbol ester, a potent protein kinase C activator that can also trigger acrosomal exocytosis, redistributes sphingosine kinase 1 to the acrosomal region. Functional assays showed that phorbol ester-induced exocytosis depends on the activation of sphingosine kinase 1. Furthermore, incorporation of 32P to sphingosine demonstrates that cells treated with the phorbol ester increase their sphingosine kinase activity that yields sphingosine 1-phosphate. We present here the first evidence indicating that human spermatozoa produce sphingosine 1-phosphate when challenged with an exocytic stimulus. These observations point to a new role of sphingosine 1-phosphate in a signaling cascade that facilitates acrosome reaction providing some clues about novel lipid molecules involved in exocytosis. PMID:20236935

  3. Sperm acrosin is responsible for the sperm binding to the egg envelope during fertilization in Japanese quail (Coturnix japonica).

    PubMed

    Sasanami, Tomohiro; Yoshizaki, Norio; Dohra, Hideo; Kubo, Hideo

    2011-08-01

    An antibody library against quail sperm plasma membrane components was established and a mAb, which strongly inhibits sperm perforations of the perivitelline membrane (PVM) was obtained from the library. The antigen molecule of the mAb showed an apparent molecular weight of 45  kDa, and was distributed both on the surface and in the acrosomal matrix of the sperm head. Periodate oxidation revealed that the epitope of the antigen includes a sugar moiety. Tandem mass spectrometry analysis of the antigen revealed that the mAb recognizes sperm acrosin. When sodium dodecyl sulfate-solubilized PVM immobilized on a polyvinylidene difluoride membrane was incubated with sperm plasma membrane lysates, the sperm acrosin was detected on the PVM immobilized on the membrane, indicating that the sperm acrosin interacts with the components of PVM. Indeed, the mAb effectively inhibited the binding of acrosome-intact sperm to the PVM. These results indicate that the 45  kDa sperm acrosin is involved in the binding of sperm to the PVM in fertilization of Japanese quail.

  4. Protein kinase C activity in boar sperm.

    PubMed

    Teijeiro, J M; Marini, P E; Bragado, M J; Garcia-Marin, L J

    2017-03-01

    Male germ cells undergo different processes within the female reproductive tract to successfully fertilize the oocyte. These processes are triggered by different extracellular stimuli leading to activation of protein phosphorylation. Protein kinase C (PKC) is a key regulatory enzyme in signal transduction mechanisms involved in many cellular processes. Studies in boar sperm demonstrated a role for PKC in the intracellular signaling involved in motility and cellular volume regulation. Experiments using phorbol 12-myristate 13-acetate (PMA) showed increases in the Serine/Threonine phosphorylation of substrates downstream of PKC in boar sperm. In order to gain knowledge about those cellular processes regulated by PKC, we evaluate the effects of PMA on boar sperm motility, lipid organization of plasma membrane, integrity of acrosome membrane and sperm agglutination. Also, we investigate the crosstalk between PKA and PKC intracellular pathways in spermatozoa from this species. The results presented here reveal a participation of PKC in sperm motility regulation and membrane fluidity changes, which is probably associated to acrosome reaction and to agglutination. Also, we show the existence of a hierarchy in the kinases pathway. Previous works on boar sperm suggest a pathway in which PKA is positioned upstream to PKC and this new results support such model.

  5. Protein tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction are enhanced in IVF media: an effect that is not associated with an increase in protein kinase A activation.

    PubMed

    Moseley, F L C; Jha, K N; Björndahl, Lars; Brewis, I A; Publicover, S J; Barratt, C L R; Lefièvre, L

    2005-07-01

    Sperm capacitation is a prerequisite for successful in vitro fertilization (IVF) and therefore a focus of sperm preparation in IVF laboratories. The technology of IVF is, therefore, potentially valuable in advancing our understanding of the molecular processes that occur during sperm capacitation. We have investigated sperm capacitation induced by a commercial IVF medium compared to that occurring in standard capacitating medium (CM) typically used in a nonclinical setting. Percoll-washed spermatozoa were resuspended in Cook Sydney IVF medium, Cook Sydney IVF sperm buffer, Earle's balanced salt medium (capacitating medium) or a modified Earle's balanced salt medium [non-capacitating medium (NCM)] for up to 120 min at 37 degrees C and, if applicable, in the presence of 5% CO2 in air. Sperm protein kinase A (PKA) activity, PKA-dependent serine/threonine phosphorylation, tyrosine phosphorylation, hyperactivation and progesterone-induced acrosome reaction were evaluated. IVF medium was shown to accelerate sperm capacitation (compared with capacitating medium) as determined by tyrosine phosphorylation, sperm hyperactivation and progesterone-induced acrosome reaction. This effect was not associated with enhanced activation of PKA or increased levels of serine/threonine phosphorylation. In contrast, IVF sperm buffer (used for sperm preparation) did not stimulate sperm capacitation when incubated for up to 90 min. We have shown that different capacitating media vary strikingly in their efficacy and that this difference reflects activation of a pathway other than the well-characterized activation of soluble adenylyl cyclase/cAMP/PKA.

  6. Effect of progesterone on acrosome reaction, hypoosmotic swelling test, and DNA stability in human spermatozoa.

    PubMed

    Contreras, H R; Roa, J; Ramirez, M A

    1999-01-01

    The association between different sperm parameters, an in vitro effect of progesterone, has not been studied satisfactorily. In this article, the effect of progesterone on acrosome reaction (AR), plasma membrane integrity, and chromatin stability has been assessed in human spermatozoa with normal morphology and motility. Semen samples were obtained by masturbation from 25 patients. Two criteria of classification were utilized in this study: high motility group and normal morphology group incubated with progesterone. The effect of progesterone on AR, plasma membrane integrity, and chromatin stability in human spermatozoa with normal morphology and motility was realized. The results suggest that only the subpopulation of spermatozoa with normal morphology is able to undergo the progesterone-induced AR. It is possible that in the reproductive female tract it takes place a high selection of sperm with chromatin stability determined and optimal plasma membrane to undergo the AR prerequisite for the fecundation.

  7. The biologically active form of the sea urchin egg receptor for sperm is a disulfide-bonded homo-multimer

    PubMed Central

    1994-01-01

    Since many cell surface receptors exist in their active form as oligomeric complexes, we have investigated the subunit composition of the biologically active sperm receptor in egg plasma membranes from Strongylocentrotus purpuratus. Electrophoretic analysis of the receptor without prior reduction of disulfide bonds revealed that the surface receptor exists in the form of a disulfide-bonded multimer, estimated to be a tetramer. These findings are in excellent agreement with the fact that the NH2-terminus of the extracellular domain of the sperm receptor is rich in cysteine residues. Studies with cross-linking agents of various length and hydrophobicity suggest that no other major protein is tightly associated with the receptor. Given the multimeric structure of the receptor, we investigated the effect of disulfide bond reduction on its biological activity. Because in quantitative bioassays fertilization was found to be inhibited by treatment of eggs with 5 mM dithiothreitol, we undertook more direct studies of the effect of reduction on properties of the receptor. First, we studied the effect of addition of isolated, pure receptor on fertilization. Whereas the non-reduced, native receptor complex inhibited fertilization in a dose- dependent manner, the reduced and alkylated receptor was inactive. Second, we tested the ability of the isolated receptor to mediate binding of acrosome-reacted sperm to polystyrene beads. Whereas beads coated with native receptor bound sperm, those containing reduced and alkylated receptor did not. Thus, these results demonstrate that the biologically active form of the sea urchin sperm receptor consists only of 350 kD subunits and that these must be linked as a multimer via disulfide bonds to produce a complex that is functional in sperm recognition and binding. PMID:8188748

  8. Chemical UV filters can affect human sperm function in a progesterone-like manner.

    PubMed

    Rehfeld, Anders; Egeberg, Dorte; Almstrup, Kristian; Holm Petersen, Jørgen; Dissing, Steen; Skakkebæk, Niels Erik

    2017-09-05

    Human sperm cell function must be precisely regulated to achieve natural fertilization. Progesterone released by the cumulus cells surrounding the egg induces a Ca2+-influx into human sperm cells via the CatSper Ca2+-channel and thereby controls sperm function. Multiple chemical UV filters have been shown to induce a Ca2+-influx through CatSper, thus mimicking the effect of progesterone on Ca2+-signaling. We hypothesized that these UV filters could also mimic the effect of progesterone on sperm function. We examined 29 UV filters allowed in sunscreens in the US and/or EU for their ability to affect acrosome reaction, penetration, hyperactivation, and viability in human sperm cells. We found that, similar to progesterone, the UV filters 4-MBC, 3-BC, Meradimate, Octisalate, BCSA, HMS and OD-PABA induced acrosome reaction, and 3-BC increased sperm penetration into a viscous medium. The capacity of the UV filters to induce acrosome reaction and increase sperm penetration was positively associated with the ability of the UV filters to induce a Ca2+-influx. None of the UV filters induced significant changes in the proportion of hyperactivated cells. In conclusion, chemical UV filters that mimic the effect of progesterone on Ca2+-signaling in human sperm cells, can similarly mimic the effect of progesterone on acrosome reaction and sperm penetration. Human exposure to these chemical UV filters may impair fertility by interfering with sperm function, e.g. through induction of premature acrosome reaction. Further studies are needed to confirm the results in vivo.

  9. Use of Sperm Enzymes to Detect Genotoxic Agents.

    DTIC Science & Technology

    1984-05-01

    film technique for detection of acrosin in single mammalian sperm, Fertile sterile 39: 548-552. Ficsor G., Oldford G. M., Loughlin K. R., Panda B. B...Genetics 77: 741-752 Ginsberg L. C., Johnson S. C., Salama M., Ficsor G. (1981): Acrosomal proteolytic assay for detection of mutagens in mammals

  10. Detection of nerve growth factor (NGF) and its specific receptor (TrkA) in ejaculated bovine sperm, and the effects of NGF on sperm function.

    PubMed

    Li, C; Sun, Y; Yi, K; Ma, Y; Sun, Y; Zhang, W; Zhou, X

    2010-12-01

    The objective was to confirm the presence of nerve growth factor (NGF) and its specific receptor, TrkA, in ejaculated bovine sperm, and to investigate the effects of NGF on specific aspects of bovine sperm function. Both TrkA transcripts and immunoreactivity typical of the translated protein were detected by RT-PCR and western blotting. However, only the NGF protein was detected in bovine sperm using western blotting, and there was no RT-PCR evidence for NGF transcripts in sperm. Using an immunofluorescent technique, NGF-immunoreactivity was localized to the sperm head and tail, whereas that of TrkA was detected in the acrosomal cap, nucleus, and tail regions When sperm were treated with exogenous NGF, both leptin secretion and sperm viability were increased (P < 0.05); moreover, the percentages of late apoptotic and dead sperm were increased (P < 0.05). However, NGF had no effects on insulin secretion, mitochondrial activity, intracellular calcium levels, or the acrosome reaction of sperm (P > 0.05). In conclusion, the presence of TrkA transcript, as well as NGF and TrkA immunoreactivity were confirmed in bovine sperm. Furthermore, exogenous NGF had significant effects on the secretion of leptin, cell viability, and sperm apoptosis. This study provided strong evidence that NGF/TrkA may have roles in regulation of sperm physiology and perhaps male fertility and infertility.

  11. Flow Cytometry Analysis Reveals That Only a Subpopulation of Mouse Sperm Undergoes Hyperpolarization During Capacitation1

    PubMed Central

    Escoffier, Jessica; Navarrete, Felipe; Haddad, Doug; Santi, Celia M.; Darszon, Alberto; Visconti, Pablo E.

    2015-01-01

    To gain fertilizing capacity, mammalian sperm should reside in the female tract for a period of time. The physiological changes that render the sperm able to fertilize are known as capacitation. Capacitation is associated with an increase in intracellular pH, an increase in intracellular calcium, and phosphorylation of different proteins. This process is also accompanied by the hyperpolarization of the sperm plasma membrane potential (Em). In the present work, we used flow cytometry to analyze changes in sperm Em during capacitation in individual cells. Our results indicate that a subpopulation of hyperpolarized mouse sperm can be clearly distinguished by sperm flow cytometry analysis. Using sperm bearing green fluorescent protein in their acrosomes, we found that this hyperpolarized subpopulation is composed of sperm with intact acrosomes. In addition, we show that the capacitation-associated hyperpolarization is blocked by high extracellular K+, by PKA inhibitors, and by SLO3 inhibitors in CD1 mouse sperm, and undetectable in Slo3 knockout mouse sperm. On the other hand, in sperm incubated in conditions that do not support capacitation, sperm membrane hyperpolarization can be induced by amiloride, high extracellular NaHCO3, and cAMP agonists. Altogether, our observations are consistent with a model in which sperm Em hyperpolarization is downstream of a cAMP-dependent pathway and is mediated by the activation of SLO3 K+ channels. PMID:25855261

  12. TRPM8, a Versatile Channel in Human Sperm

    PubMed Central

    Ocampo, Ana Y.; Serrano, Carmen J.; Castellano, Laura E.; Hernández-González, Enrique O.; Chirinos, Mayel; Larrea, Fernando; Beltrán, Carmen; Treviño, Claudia L.

    2009-01-01

    Background The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca2+ ([Ca2+]i). Ca2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. Principal Findings Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 µM) and 80% by BCTC (1.6 µM). Activation of TRPM8 either by temperature or menthol induced [Ca2+]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 µM) and BCTC (1.6 µM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. Conclusions This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis. PMID:19582168

  13. Puma (Puma concolor) epididymal sperm morphometry.

    PubMed

    Cucho, Hernán; Alarcón, Virgilio; Ordóñez, César; Ampuero, Enrique; Meza, Aydee; Soler, Carles

    2016-01-01

    The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species.

  14. Puma (Puma concolor) epididymal sperm morphometry

    PubMed Central

    Cucho, Hernán; Alarcón, Virgilio; Ordóñez, César; Ampuero, Enrique; Meza, Aydee; Soler, Carles

    2016-01-01

    The Andean puma (Puma concolor) has not been widely studied, particularly in reference to its semen characteristics. The aim of the present study was to define the morphometry of puma sperm heads and classify their subpopulations by cluster analysis. Samples were recovered postmortem from two epididymides from one animal and prepared for morphological observation after staining with the Hemacolor kit. Morphometric data were obtained from 581 spermatozoa using a CASA-Morph system, rendering 13 morphometric parameters. The principal component (PC) analysis was performed followed by cluster analysis for the establishment of subpopulations. Two PC components were obtained, the first related to size and the second to shape. Three subpopulations were observed, corresponding to elongated and intermediate-size sperm heads and acrosomes, to large heads with large acrosomes, and to small heads with short acrosomes. In conclusion, puma spermatozoa showed no uniform sperm morphology but three clear subpopulations. These results should be used for future work in the establishment of an adequate germplasm bank of this species. PMID:27678466

  15. A phosphodiesterase type-5 inhibitor, sildenafil, induces sperm capacitation and penetration into porcine oocytes in a chemically defined medium.

    PubMed

    Ioki, Sumire; Wu, Qing-Shan; Takayama, Osamu; Motohashi, Hideyuki H; Wakai, Takuya; Funahashi, Hiroaki

    2016-02-01

    The present study was undertaken to determine the effect of a phosphodiesterase (PDE) type-5 (cyclic guanosine monophosphate-specific) inhibitor, sildenafil, on capacitation and penetration of boar spermatozoa in a basic chemically defined medium (adenosine- and theophylline-free PGM-tac4). When ejaculated spermatozoa were cultured for 90 minutes in the absence or presence of sildenafil at 2.5 mM, the inhibitor significantly increased the percentage of capacitated/acrosome-reacted spermatozoa, as a result of the chlortetracycline assay. When fresh spermatozoa were co-cultured with oocytes in the presence of sildenafil at a different concentration (0, 2.5, 25, or 250 μM), higher sildenafil concentrations (25 and 250 μM) significantly resulted in higher sperm penetration rates. When oocytes matured in vitro were co-cultured with spermatozoa in the presence of 25 μM sildenafil or 25 mM caffeine benzoate for 8 hours, the incidence of penetrated oocytes did not differ between two groups, whereas the incidence of monospermic oocytes in penetrated one was significantly higher in the presence of sildenafil. Immunocytochemical analysis reported the presence of PDE type-5 on the acrosome region of boar spermatozoa. These results report that regulation of cyclic guanosine monophosphate-specific PDE type-5 by sildenafil somehow can increase the penetrability of boar spermatozoa in vitro.

  16. Characterization of functional variables in epididymal alpaca (Vicugna pacos) sperm using imaging flow cytometry.

    PubMed

    Santiani, Alexei; Ugarelli, Alejandra; Evangelista-Vargas, Shirley

    2016-10-01

    Epididymal alpaca sperm represent an alternative model for the study of alpaca semen. The objective of this study was to characterize the normal values of some functional variables in epididymal alpaca sperm using imaging flow cytometry. Alpaca testicles (n=150) were processed and sperm were recovered from the cauda epididymides. Only 76 samples with acceptable motility and sperm count were considered for assessment by imaging flow cytometry. Acrosome integrity and integrity/viability were assessed by FITC-PSA/PI and FITC-PNA/PI. Mitochondrial membrane potential (MMP) was assessed by MitoTracker CMXRos and MitoTracker Deep Red FM. Lipid peroxidation was evaluated using BODIPY 581/591 C11. Results show that the mean values for acrosome-intact sperm were 95.03±6.39% and 93.34±7.96%, using FITC-PSA and FITC-PNA, respectively. The mean values for acrosome-intact viable sperm were 60.58±12.12% with FITC-PSA/PI and 58.81±12.94% with FITC-PNA/PI. Greater MMP was detected in 65.03±15.92% and 59.52±19.19%, using MitoTracker CMXRos and MitoTracker Deep Red FM, respectively. Lipid peroxidation was 0.84±0.95%. Evaluation of acrosome-intact and acrosome-intact viable sperm with FITC-PSA/PI compared with. FITC-PNA/PI or MMP with MitoTracker CMXRos compared with MitoTracker Deep Red FM were correlated (P<0.05). The MMP using MitoTracker CMXRos was the only variable correlated (P<0.05) with sperm motility (r=0.3979). This report provides a basis for future research related to alpaca semen using the epididymal sperm model.

  17. Effect of butylated hydroxytoluene on dog sperm longevity in chilling storage and cryopreservation.

    PubMed

    Sahashi, Yu; Otsuki, Tsubasa; Higaki, Shogo; Nagano, Masashi; Yamashita, Yasuhisa; Hishinuma, Mitsugu

    2011-07-01

    This study evaluated the effect of butylated hydroxytoluene (BHT), a lipid-soluble antioxidant, on dog sperm in chilling storage and cryopreservation. In Experiment 1, 0.2, 0.4, 0.8 and 1.6 mM BHT were added to egg yolk Tris extender (EYT), and sperm were stored at 4°C for 96 hr. Sperm motility, viability, acrosomal integrity and morphological abnormality in the BHT treatment groups were not different from those of the control (0 mM BHT). In Experiment 2, the effect of BHT in EYT containing 0.75% Equex STM paste and 5% glycerol on survivability of cryopreserved sperm was examined after culture at 39°C for 3 hr. Sperm motility, viability and acrosomal integrity in the 0.2 to 0.8 mM BHT treatment groups were not different from those of the control. However, sperm motility, viability and acrosomal integrity decreased when 1.6 mM BHT was added to the extender (P<0.05). In conclusion, supplementation of the extender with 0.2 to 0.8 mM BHT did not affect characteristics of dog sperm in chilling storage and cryopreservation. Supplementation of 1.6 mM BHT did not affect characteristics of chilled sperm but impaired longevity of cryopreserved sperm in the dog.

  18. Metabolic activity of sperm cells: correlation with sperm cell concentration, viability and motility in the rabbit.

    PubMed

    Sabés-Alsina, Maria; Planell, Núria; Gil, Sílvia; Tallo-Parra, Oriol; Maya-Soriano, Maria José; Taberner, Ester; Piles, Miriam; Sabés, Manel; Lopez-Bejar, Manel

    2016-10-01

    The resazurin reduction test (RRT) is a useful technique to assess the metabolic rate of sperm cells. RRT depends on the ability of metabolically active cells to reduce the non-fluorescent dye resazurin to the fluorescent resorufin. The aim of this study was to develop a vital fluorometric method to evaluate metabolic activity of rabbit sperm cells. Twenty-five rabbit males were included in the study. Viability and morphology, motility and metabolic activity were evaluated using an eosin-nigrosin staining, a computer-assisted semen analysis (CASA) and the RRT, respectively. Spearman rank correlation analysis was used to determine the correlation between RRT and semen parameters. After evaluation, a concentration of 10 × 106 sperm cells/ml was selected for further experiments with RRT. No significant correlation was found between the RRT results and the motility parameters. However, after RRT a significant positive correlation between relative fluorescence units and the percentage of alive spermatozoa (r = 0.62; P = 0.001) and a negative one with the percentage of sperm cells with acrosomic abnormalities (r = -0.45; P < 0.05) were detected. The vital assessment of metabolic rate of sperm cells by RRT could provide more information about semen quality than other routine semen analysis, correlating with sperm viability and acrosome status information.

  19. Intracellular pH in Sperm Physiology

    PubMed Central

    Nishigaki, Takuya; José, Omar; González-Cota, Ana Laura; Romero, Francisco; Treviño, Claudia L.; Darszon, Alberto

    2014-01-01

    Intracellular pH (pHi) regulation is essential for cell function. Notably, several unique sperm ion transporters and enzymes whose elimination causes infertility are either pHi dependent or somehow related to pHi regulation. Amongst them are: CatSper, a Ca2+ channel; Slo3, a K+ channel; the sperm-specific Na+/H+ exchanger and the soluble adenylyl cyclase. It is thus clear that pHi regulation is of the utmost importance for sperm physiology. This review briefly summarizes the key components involved in pHi regulation, their characteristics and participation in fundamental sperm functions such as motility, maturation and the acrosome reaction. PMID:24887564

  20. A comparative study of the morphometry of sperm head components in cattle, sheep, and pigs with a computer-assisted fluorescence method

    PubMed Central

    Yániz, Jesús L; Capistrós, Sara; Vicente-Fiel, Sandra; Hidalgo, Carlos O; Santolaria, Pilar

    2016-01-01

    The aim of this study was to compare the sperm nuclear and acrosomal morphometry of three species of domestic artiodactyls; cattle (Bos taurus), sheep (Ovis aries), and pigs (Sus scrofa). Semen smears of twenty ejaculates from each species were fixed and labeled with a propidium iodide-Pisum sativum agglutinin (PI/PSA) combination. Digital images of the sperm nucleus, acrosome, and whole sperm head were captured and analyzed. The use of the PI/PSA combination and CASA-Morph fluorescence-based method allowed the capture, morphometric analysis, and differentiation of most sperm nuclei, acrosomes and whole heads, and the assessment of acrosomal integrity with a high precision in the three species studied. For the size of the head and nuclear area, the relationship between the three species may be summarized as bull > ram > boar. However, for the other morphometric parameters (length, width, and perimeter), there were differences in the relationships between species for sperm nuclei and whole sperm heads. Bull sperm acrosomes were clearly smaller than those in the other species studied and covered a smaller proportion of the sperm head. The acrosomal morphology, small in the bull, large and broad in the sheep, and large, long, and with a pronounced equatorial segment curve in the boar, was species-characteristic. It was concluded that there are clear variations in the size and shape of the sperm head components between the three species studied, the acrosome being the structure showing the most variability, allowing a clear distinction of the spermatozoa of each species. PMID:27624987

  1. ICSI choreography: fate of sperm structures after monospermic rhesus ICSI and first cell cycle implications.

    PubMed

    Ramalho-Santos, J; Sutovsky, P; Simerly, C; Oko, R; Wessel, G M; Hewitson, L; Schatten, G

    2000-12-01

    We have dissected the initial stages of fertilization by intracytoplasmic sperm injection of single spermatozoa into prime oocytes from fertile rhesus monkeys (Macaca mulatta). DNA decondensation was delayed at the apical portion of the sperm head. It is possible that this asynchronous male DNA decondensation could be related to the persistence of the sperm acrosome and perinuclear theca after injection. However, incomplete male pronuclear formation did not prevent sperm aster formation, microtubule nucleation and pronuclear apposition. In contrast, DNA synthesis was delayed in both pronuclei until the sperm chromatin fully decondensed, indicating that male pronuclear formation constitutes an important checkpoint during the first embryonic cell cycle.

  2. MOLECULAR ARCHITECTURE OF THE HUMAN SPERM IZUMO1 AND EGG JUNO FERTILIZATION COMPLEX

    PubMed Central

    Aydin, Halil; Sultana, Azmiri; Li, Sheng; Thavalingam, Annoj; Lee, Jeffrey E.

    2017-01-01

    Fertilization is an essential biological process in sexual reproduction and comprises a series of molecular interactions between the sperm and egg1,2. The fusion of haploid spermatozoon and oocyte is the culminating event in mammalian fertilization, enabling the creation of a new genetically distinct diploid organism3,4. The merger of two gametes is achieved through a two-step mechanism where the sperm Izumo1 on the equatorial segment of the acrosome-reacted sperm recognizes its receptor Juno, on the egg surface4–6. This is followed by the fusion of two plasma membranes. Izumo1 and Juno proteins are indispensable for fertilization as constitutive knockout of either Izumo1 or Juno result in mice that are healthy but infertile5,6. Despite their central importance in reproductive medicine, the molecular architectures and the details of their functional roles in fertilization are not known. Here, we present the crystal structures of the human Izumo1 and Juno in unbound and bound conformations. The human Izumo1 structure exhibits a distinct boomerang shape and provides the first structural insights into the Izumo family of proteins7. Human Izumo1 forms a high-affinity complex with Juno and undergoes a major conformational change within its N-terminal domain upon binding to the egg-surface receptor. Our results provide new insights into the molecular basis of sperm-egg recognition, cross-species fertilization, and barrier to polyspermy, thus promising benefits for the rational development of novel non-hormonal contraceptives and fertility treatments for humans and other species of mammals. PMID:27309818

  3. Fusion of artificial membranes with mammalian spermatozoa. Specific involvement of the equatorial segment after acrosome reaction.

    PubMed

    Arts, E G; Kuiken, J; Jager, S; Hoekstra, D

    1993-11-01

    The fusogenic properties of bovine and human spermatozoa membranes were investigated, using phospholipid bilayers (liposomes) as target membranes. Fusion was monitored by following lipid mixing, as revealed by an assay based on resonance-energy transfer. In addition, fusion was visualized by fluorescence microscopy, using fluorescent lipid vesicles. Cryopreserved bovine sperm fused with liposomes before induction of the acrosome reaction, fluorescence being located in essentially all spermatozoa membrane domains. Fresh bovine and human spermatozoa fused with liposomes only after the induction of the acrosome reaction, as triggered by calcium ionophore A23187 or zonae pellucidae (proteins), while the fluorescence distribution was mainly restricted to the equatorial segment (ES). However, with spermatozoa that had undergone a freeze/thawing cycle, domains other than ES also became labeled. Hence, the redistribution of the lipid probes over the entire membrane occurring during lipid mixing with cryopreserved bovine sperm is probably related to membrane perturbations caused by long-term cryopreservation. Fusion with liposomes was governed by spermatozoa factors and required the presence of acidic phospholipids like cardiolipin and phosphatidylserine in the liposomal bilayer. Incorporation of the zwitterionic lipid phosphatidylcholine in the vesicles inhibited the fusion reaction. Fusion was pH dependent. The results indicate that the ES is the primary domain of spermatozoa membranes that harbours the fusogenic capacity of sperm. Liposomes appear a valuable tool in further characterizing the properties of this domain, which has been claimed [Yanagimachi, R. (1988) in The physiology of reproduction (Knobil, E. & Neill, J., eds) pp. 135-185, Raven Press, New York] to represent the putative, initial fusion site for the oocyte.

  4. Effect of cryoprotective diluent and method of freeze-thawing on survival and acrosomal integrity of ram spermatozoa.

    PubMed

    Pontbriand, D; Howard, J G; Schiewe, M C; Stuart, L D; Wildt, D E

    1989-08-01

    A multifactorial study analyzed the effects of freezing method, cryoprotective diluent, semen to diluent ratio, and thawing velocity on post-thaw motility, progressive status, and acrosomal integrity of ram spermatozoa. Although semen to diluent ratio (1:3 vs 1:6, v/v) had no effect (P greater than 0.05), overall post-thaw spermatozoal viability was highly dependent on freezing method and cryoprotectant. Improved results were obtained by freezing semen in 0.5-ml French straws compared to dry ice pelleting. Manually freezing straws 5 cm above liquid nitrogen (LN2) was comparable to cooling straws in an automated, programmable LN2 unit. Of the two cryoprotective diluents tested, BF5F (containing the surfactant component sodium and triethanolamine lauryl sulfate) yielded approximately 50% fewer (P less than 0.05) spermatozoa with loose acrosomal caps compared to TEST. Thawing straws in a water bath at a higher velocity (60 degrees C for 8 sec) had no effect (P greater than 0.05) on spermatozoal motility, progressive status ratings, or acrosomal integrity when compared to a lower rate (37 degrees C for 20 sec). For the TEST group, thawing pellets in a dry, glass culture tube promoted (P less than 0.05) percentage sperm motility at 3 and 6 hr post-thawing, but for BF5F diluted semen this approach decreased the % of spermatozoa with normal apical ridges. The results suggest that the poor fertility rates often experienced using thawed ram semen likely result not only from reduced sperm motility, but also from compromised ultrastructural integrity. This damage is expressed by an increased loosening of the acrosomal cap, a factor which appears insensitive to freezing method but markedly influenced by the cryoprotective properties of the diluents tested.

  5. Protective effect of hyaluronic acid on cryopreserved boar sperm.

    PubMed

    Qian, Li; Yu, Sijiu; Zhou, Yan

    2016-06-01

    This study aimed to evaluate the effects of supplementing freezing and thawing media with hyaluronic acid (HA) on the quality parameters of frozen-thawed boar spermatozoa. Boar semen samples were collected from seven mature Yorkshire boars once a week using the gloved hand technique; these samples were frozen-thawed in the extender with added HA. Boar sperm was cryopreserved in the extender with HA added at concentrations of 0 (used as control), 4, 6, 8, 8 and 12mg/L, and their effects on the quality of frozen-thawed boar sperm were evaluated. HA addition to the extender significantly improved sperm motility, sperm membrane integrity, mitochondrial activity, acrosomal integrity, superoxide dismutase and catalase activity, but decreased sperm malondialdehyde level (p<0.05). Therefore, HA could be a promising cryoprotectant for boar sperm.

  6. Impact of kudzu and puerarin on sperm function.

    PubMed

    Gray, Sandra L; Lackey, Brett R; Boone, William R

    2015-06-01

    The goal of this study was to investigate the impact of kudzu (Pueraria mirifica) and the isoflavone puerarin in functional toxicological tests on spermatozoa and to assess the affinity of extracts and pure isoflavones for estrogen receptor (ER)-alpha and -beta (ERα, ERβ) in receptor binding assays. Capacitation, acrosome reaction and chromatin decondensation in spermatozoa were analyzed using microscopic analysis. Kudzu, but not puerarin, reduced motility of sperm. Puerarin reduced the percent spontaneous acrosome reaction in spermatozoa. The pathways used by kudzu that affect sperm function are not fully mirrored by puerarin. Puerarin, kudzu and its other phytoestrogenic components displayed preferential affinity for ERβ, however the diverse effects of kudzu and puerarin on sperm function implicate the involvement of multiple signaling systems.

  7. Versatile Action of Picomolar Gradients of Progesterone on Different Sperm Subpopulations

    PubMed Central

    Uñates, Diego Rafael; Guidobaldi, Héctor Alejandro; Gatica, Laura Virginia; Cubilla, Marisa Angélica; Teves, María Eugenia; Moreno, Ayelén; Giojalas, Laura Cecilia

    2014-01-01

    High step concentrations of progesterone may stimulate various sperm physiological processes, such as priming and the acrosome reaction. However, approaching the egg, spermatozoa face increasing concentrations of the hormone, as it is secreted by the cumulus cells and then passively diffuses along the cumulus matrix and beyond. In this context, several questions arise: are spermatozoa sensitive to the steroid gradients as they undergo priming and the acrosome reaction? If so, what are the functional gradual concentrations of progesterone? Do spermatozoa in different physiological states respond differentially to steroid gradients? To answer these questions, spermatozoa were confronted with progesterone gradients generated by different hormone concentrations (1 pM to 100 µM). Brief exposure to a 10 pM progesterone gradient stimulated priming for the acrosome reaction in one sperm subpopulation, and simultaneously induced the acrosome reaction in a different sperm subpopulation. This effect was not observed in non-capacitated cells or when progesterone was homogeneously distributed. The results suggest a versatile role of the gradual distribution of very low doses of progesterone, which selectively stimulate the priming and the acrosome reaction in different sperm subpopulations. PMID:24614230

  8. P38 participates in spermatogenesis and acrosome reaction prior to fertilization in Chinese mitten crab Eriocheir sinensis.

    PubMed

    Zhu, Ming; Sun, Wen-Juan; Wang, Yuan-Li; Li, Qing; Yang, Hong-Dan; Duan, Ze-Lin; He, Lin; Wang, Qun

    2015-04-01

    P38 mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine protein kinases that play important roles in cellular responses to inflammatory cytokines and environmental stresses. These kinases are involved in controlling cell division, differentiation and death in mammalian testes and therefore are critical to spermatogenesis. To explore their functions in male reproduction of Chinese mitten crabs, Eriocheir sinensis p38 (Es-p38) protein expression was determined in different tissues including testes at different developmental stages by Western blot. Es-p38 was expressed in various tissues, with higher levels in the heart, stomach, gills and testes. Total Es-p38 protein levels increased gradually during spermatogenesis, but phosphorylated Es-p38 was much higher in the spermatid (August-October) than the spermatocyte (July-August) and sperm (October-January) stages. Trypan blue staining and hematoxylin/eosin staining were both used to detect sperm motility and changes in sperm morphology during the acrosome reaction (AR) induced by pre-incubation with A23187 in vitro, activated Es-p38 proteins detected by fluorescent microscopy were translocated gradually to nuclear and apical cap regions, accumulating at the anterior of the acrosomal tubule. The results suggest the involvement of p38 MAPK in spermatogenesis and the AR in E. sinensis.

  9. Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

    PubMed

    Zimmerman, Shawn W; Manandhar, Gaurishankar; Yi, Young-Joo; Gupta, Satish K; Sutovsky, Miriam; Odhiambo, John F; Powell, Michael D; Miller, David J; Sutovsky, Peter

    2011-02-23

    Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced

  10. Effects of liquid preservation of sperm on their ability to activate oocytes and initiate preimplantational development after intracytoplasmic sperm injection in the pig.

    PubMed

    Binh, N T; Van Thuan, N; Miyake, M

    2009-06-01

    The objective of this study was to clarify the effects of liquid preservation conditions on the ability of pig sperm to activate oocytes, form a male pronucleus, and initiate preimplantational development of embryos after intracytoplasmic sperm injection (ICSI). Porcine ejaculates were preserved at 4, 14, and 24 degrees C for up to 48h, and then damage to the plasma membrane, morphologic changes of the acrosome, and the amount of phospholipase Czeta (PLCzeta) in the sperm were assessed by SYBR-14/propidium iodide staining, fluorescein isothiocyanate-conjugated peanut agglutinin staining, indirect immunofluorescence, and Western blots, respectively. The proportion of sperm with a disintegrated plasma membrane or damaged acrosome increased in all samples as the duration of preservation increased, although the time courses of the increases varied among preservation temperatures. The immunolocalization and immunoreactivity of PLCzeta in the sperm showed its reduction concurrent with disintegration of the plasma membrane and acrosome. Rates of oocyte activation, male-pronuclear formation, and blastocyst formation after ICSI using sperm preserved for 18h at 24 degrees C (78%, 62%, and 35%, respectively) and for 48h at 14 degrees C (63%, 53%, and 28%, respectively) were significantly higher than those of any other sperm sample. We concluded that the damage to the plasma membrane and acrosome, and a sufficient amount of PLCzeta in the sperm head, enhanced successful oocyte activation, fertilization, and early development of the oocytes after ICSI. Moreover, we inferred that appropriate liquid preservation of sperm improved the efficiency of blastocyst production in vitro after ICSI in pigs.

  11. Roles of the zona pellucida and functional exposure of the sperm-egg fusion factor 'IZUMO' during in vitro fertilization in pigs.

    PubMed

    Tanihara, Fuminori; Nakai, Michiko; Men, Nguyen Thi; Kato, Noriko; Kaneko, Hiroyuki; Noguchi, Junko; Otoi, Takeshige; Kikuchi, Kazuhiro

    2014-04-01

    The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm-egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP-intact and ZP-free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44-0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti-IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti-IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP-free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.

  12. Centrifugation on Percoll density gradient enhances motility, membrane integrity and in vitro fertilizing ability of frozen-thawed boar sperm.

    PubMed

    Noguchi, Michiko; Yoshioka, Koji; Hikono, Hirokazu; Iwagami, Gentaro; Suzuki, Chie; Kikuchi, Kazuhiro

    2015-02-01

    The effects of Percoll density gradient centrifugation on sperm quality, in vitro fertilizability and developmental capacity of frozen-thawed boar sperm were evaluated. Two-step density gradient centrifugation by Percoll enhanced significantly the motility parameters of sperm compared with a simple centrifugation procedure. Percentages of motile sperm and sperm with intact plasma and acrosome membranes after Percoll separation were significantly greater than those after simple centrifugation. The rates of penetration, cleavage and blastocyst formation after in vitro fertilization were significantly improved by Percoll separation compared with simple centrifugation and were influenced positively by the intactness of sperm head membranes, but not any sperm motility parameters. However, insemination with increased concentrations of sperm prepared by Percoll gradient centrifugation did not improve the success of fertilization and embryo development in vitro. Our results indicate that the integrity of sperm head membranes after Percoll separation is important for successful embryo development in vitro, more so than sperm motility.

  13. Novel and traditional traits of frozen-thawed porcine sperm related to in vitro fertilization success.

    PubMed

    Daigneault, Bradford W; McNamara, Kelli A; Purdy, Phillip H; Krisher, Rebecca L; Knox, Robert V; Miller, David J

    2014-07-15

    Cryopreserved semen allows the use of single ejaculates for repeated analyses, potentially improving IVF consistency by eliminating interejaculate variability observed with fresh semen. However, the freezing and thawing processes result in compromised sperm function and IVF success. Semen samples are often screened for motility before use for IVF. Samples that are below a designated motility threshold may be discarded. Our objectives were to determine if post-thaw sperm motility, other traits that may be indicative of sperm function, or a novel assay of oviduct binding were related to IVF success. Semen from 16 boars was cooled to 15 °C for overnight shipment before cryopreservation. Semen was thawed and motility was recorded microscopically and confirmed using computer-automated sperm assessment. Each sample was tested by IVF in two to three independent replicates. Regression and correlation analyses were employed to determine the interrelationships between sperm traits and the relationships between post-thaw motility, sperm-oviduct binding and IVF outcomes. Among the sperm traits examined, sperm acrosome integrity was negatively correlated with post-thaw motility (r(2) = 0.64) but not with IVF results. The number of sperm bound to oviduct aggregates was correlated with IVF polyspermy rates (r(2) = 0.62, P < 0.05) but less with overall IVF rates (r(2) = 0.31, P > 0.10). There was some relationship of post-thaw motility with IVF monospermic fertilization (P = 0.06, r(2) = 0.08) but not to other IVF outcomes. Our results indicate that post-thaw motility of frozen-thawed boar sperm is strongly related to acrosome integrity but has limited use for predicting IVF success. The number of sperm bound to oviduct cells was related to IVF polyspermy rates and may be more indicative of in vitro sperm function than traditional sperm motility and acrosome status evaluation.

  14. Effects of Synthetic Serum Supplementation in Sperm Preparation Media on Sperm Capacitation and Function Test Results

    PubMed Central

    Shih, Ying-Fu; Tzeng, Shu-Ling; Huang, Chun-Chia

    2016-01-01

    Albumin supplementation of culture media induces sperm capacitation in assisted reproduction technique cycles. Synthetic serum supplementation is clinically used to replace albumin for preventing transmission of infectious agents. However, the effects of synthetic serum supplementation on sperm capacitation have rarely been investigated. Spermatozoa from 30 men with normal basic semen analysis results were collected, divided into five aliquots, and cultured in capacitating conditions in four combinations of two synthetic serum supplements, serum substitute supplement (SSS) and serum protein substitute (SPS), and two fertilization media, Quinns Advantage™ Fertilization (QF) and human tubular fluid (HTF) media. Reactive oxygen species (ROS) levels in spermatozoa were measured through chemiluminescence. Furthermore, acrosome reaction and western blotting for tyrosine phosphorylation were used to evaluate sperm capacitation. HTF+SSS had significantly higher ROS levels than QF+SPS did (11,725 ± 1,172 versus 6,278 ± 864 relative light units). In addition, the spermatozoa cultured in QF+SPS had lower motility, acrosome reaction rates, and tyrosine phosphorylation levels compared with those cultured in HTF+SSS. In conclusion, the effects of synthetic serum supplementation on sperm capacitation varied according to the combination of media. These differences may lead to variations in spermatozoon ROS levels, thus affecting sperm function test results. PMID:27413417

  15. Sperm characterization and identification of sperm sub-populations in ejaculates from pampas deer (Ozotoceros bezoarticus).

    PubMed

    Beracochea, F; Gil, J; Sestelo, A; Garde, J J; Santiago-Moreno, J; Fumagalli, F; Ungerfeld, R

    2014-10-01

    Pampas deer (Ozotoceros bezoarticus) is a native endangered species. Knowledge of the basic spermiogram characteristics and the morphometric descriptors is necessary to effectively develop sperm cryopreservation. In other species, sperm sub-population is related to sperm cryo-resistance. The objective was to provide a general description of the sperm, including sperm head morphometric descriptors, its repeatability, and the existence of sperm sub-populations. Sperm were obtained from adult males by electroejaculation during the breeding season. The motility score was 3.4 ± 0.2 (mean ± SEM) and progressive motility was 59.4 ± 3.7%. Ejaculated volume was 413.9 ± 51.0 μl, the total number of sperm ejaculated was 321.2 ± 55.4 × 10(6). Also, 63.3 ± 3.1% of the sperm were morphologically abnormal and 23.7 ± 2.3% had acrosome damage. The sperm head length was 7.6 ± 0.01 μm, width 4.4 ± 0.01 μm, area 28.1 ± 0.07 μm(2) and the perimeter was 21.9 ± 0.04 μm. There was a positive relationship among morphometric descriptors and the motility score, overall motility and progressive motility. Also length (P=0.011), width (P=0.003), area (P=0.006) and perimeter (P=0.009) of sperm head obtained in two different collections were positively related. Overall, the low concentration, volume, overall quality and abnormal morphology, and wide variation of these variables may be a limitation for the development of sperm cryopreserved banks. There were three sperm sub-populations with different morphometric characteristics. The morphometric descriptors are maintained similarly among different collections. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Viability and acrosome staining of stallion spermatozoa by Chicago sky blue and Giemsa.

    PubMed

    Kútvölgyi, G; Stefler, J; Kovács, A

    2006-01-01

    A simple trypan blue-neutral red-Giemsa staining procedure for simultaneous evaluation of acrosome, sperm head, and tail membrane integrity and morphology has been used to evaluate equine spermatozoa. Some special characteristics and problems have arisen in evaluating stallion semen. One problem was the differentiation of intact vs. damaged sperm tails primarily in frozen and thawed samples. After freezing and thawing, a high percentage of spermatozoa with an unstained head and stained tail were observed. These cells are considered immotile. Therefore, unambiguous differentiation of intact vs. damaged sperm tail membrane is very important for evaluating semen quality. The aim of our study was to develop a method especially for stallion sperm to distinguish more accurately the different cell types. We compared Chicago sky blue 6B (CSB) to trypan blue (TB) for viability staining. CSB/Giemsa staining showed good repeatability and agreement with TB/Giemsa measurements. For densitometry analysis, individual digital images were taken from smears stained by CSB/Giemsa and by TB/Giemsa. A red-green-blue (RGB) histogram for each area of spermatozoa was drawn. Differences of means of RGB values of live vs. dead tails and separate live vs. dead heads from each photo were used to compare the two staining procedures. CSB produced similar live/dead sperm head differentiation and better tail differentiation. TB can be replaced by CSB and this results in more reliable evaluation. After staining with 0.16% CSB and 4 min fixation, 2-4 h Giemsa staining at 25-40 degrees C is recommended for stallion semen.

  17. Increased chromatin fragmentation and reduced acrosome integrity in spermatozoa of red deer from lead polluted sites.

    PubMed

    Castellanos, Pilar; del Olmo, Enrique; Fernández-Santos, M Rocío; Rodríguez-Estival, Jaime; Garde, J Julián; Mateo, Rafael

    2015-02-01

    Vertebrates are constantly exposed to a diffuse pollution of heavy metals existing in the environment, but in some cases, the proximity to emission sources like mining activity increases the risk of developing adverse effects of these pollutants. Here we have studied lead (Pb) levels in spermatozoa and testis, and chromatin damage and levels of endogenous antioxidant activity in spermatozoa of red deer (Cervus elaphus) from a Pb mining area (n=37) and a control area (n=26). Deer from the Pb-polluted area showed higher Pb levels in testis parenchyma, epididymal cauda and spermatozoa, lower values of acrosome integrity, higher activity of glutathione peroxidase (GPx) and higher values of DNA fragmentation (X-DFI) and stainability (HDS) in sperm than in the control area. These results indicate that mining pollution can produce damage on chromatin and membrane spermatozoa in wildlife. The study of chromatin fragmentation has not been studied before in spermatozoa of wildlife species, and the sperm chromatin structure assay (SCSA) has been revealed as a successful tool for this purpose in species in which the amount of sperm that can be collected is very limited. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Effect of storage of domestic cat (Felis catus) epididymides at 5 degrees C on sperm quality and cryopreservation.

    PubMed

    Gañán, N; Gomendio, M; Roldan, E R S

    2009-12-01

    Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91+/-2.02, 48.21+/-1.47, and 43.03+/-1.32) and after thawing (51.81+/-3.02, 41.90+/-2.14, and 42.35+/-1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33+/-1.38% before and 52.50+/-1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22+/-1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI=39.17+/-2.76 and 45.00+/-2.65, respectively) and the percentage of intact acrosomes (45.76+/-4.91% and 60.67+/-3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.

  19. Improving sperm banking efficiency in endangered species through the use of a sperm selection method in brown bear (Ursus arctos) thawed sperm.

    PubMed

    Anel-Lopez, L; Ortega-Ferrusola, C; Álvarez, M; Borragán, S; Chamorro, C; Peña, F J; Morrell, J; Anel, L; de Paz, P

    2017-06-26

    Sperm selection methods such as Single Layer Centrifugation (SLC) have been demonstrated to be a useful tool to improve the quality of sperm samples and therefore to increase the efficiency of other artificial reproductive techniques in several species. This procedure could help to improve the quality of genetic resource banks, which is essential for endangered species. In contrast, these sperm selection methods are optimized and focused on farm animals, where the recovery task is not as important as in endangered species because of their higher sperm availability. The aim of this study was to evaluate two centrifugation methods (300 x g/20 min and 600 x g/10 min) and three concentrations of SLC media (Androcoll-Bear -80, 65 and 50%) to optimise the procedure in order to recover as many sperm with the highest quality as possible. Sperm morphology could be important in the hydrodynamic relationship between the cell and centrifugation medium and thus the effect of sperm head morphometry on sperm yield and its hydrodynamic relationship were studied. The samples selected with Androcoll-Bear 65% showed a very good yield (53.1 ± 2.9) although the yield from Androcoll-Bear 80% was lower (19.3 ± 3.3). The latter showed higher values of motility than the control immediately after post-thawing selection. However, both concentrations of colloid (65 and 80%) showed higher values of viable sperm and viable sperm with intact acrosome than the control. After an incubation of 2 h at 37 °C, the samples from Androcoll-Bear 80% had higher kinematics and proportion of viable sperm with intact acrosome. In the morphometric analysis, the sperm selected by the Androcoll-Bear 80% showed a head with a bigger area which was more elongated than the sperm from other treatments. We conclude that sperm selection with Androcoll-Bear at either 65% or 80% is a suitable technique that allows a sperm population with better quality than the initial sample to be obtained. We recommend the

  20. The hypo-osmotic swelling test for evaluation of sperm membrane integrity.

    PubMed

    Ramu, Sivakumar; Jeyendran, Rajasingam S

    2013-01-01

    A functional membrane is requisite for the fertilizing ability of spermatozoa, as it plays an integral role in sperm capacitation, acrosome reaction, and binding of the spermatozoon to the egg surface. The hypo-osmotic swelling (HOS) test evaluates the functional integrity of the sperm's plasma membrane and also serves as a useful indicator of fertility potential of sperm. The HOS test predicts membrane integrity by determining the ability of the sperm membrane to maintain equilibrium between the sperm cell and its environment. Influx of the fluid due to hypo-osmotic stress causes the sperm tail to coil and balloon or "swell." A higher percentage of swollen sperm indicates the presence of sperm having a functional and intact plasma membrane. Here, we present the detailed protocol for performing the HOS test and explain the results for interpretation.

  1. How is plasminogen/plasmin system contributing to regulate sperm entry into the oocyte?

    PubMed

    Grullón, Luis A; Gadea, Joaquín; Mondéjar, Irene; Matás, Carmen; Romar, Raquel; Coy, Pilar

    2013-09-01

    Plasminogen is present in the oviduct, on the zona pellucida (ZP) and on oolemma, and reduces the number of sperm penetrating the oocyte during in vitro fertilization in pig and cow. It is unknown how this reduction occurs. We tested whether plasminogen (1) changed the ZP resistance to enzymatic digestion thus making the passage of the spermatozoa across it difficult; (2) reduced the sperm functionality, assessed by sperm viability, motility, spontaneous acrosome reaction and membrane lipid disorder; or (3) affected the sperm-ZP binding before or after sperm-ZP interaction. The mechanism by which plasminogen/plasmin system contributes to regulate sperm entry into the oocyte is not inducing a ZP hardening or a decrease in sperm functionality but detaching more than 50% of sperm bound to the ZP. It is suggested that the fertilizing spermatozoon activates plasminogen into plasmin at the oocyte surface and that plasmin removes additional spermatozoa attached to the ZP.

  2. Role of C-type natriuretic peptide in the function of normal human sperm

    PubMed Central

    Xia, Hui; Chen, Yao; Wu, Ke-Jia; Zhao, Hu; Xiong, Cheng-Liang; Huang, Dong-Hui

    2016-01-01

    C-type natriuretic peptide (CNP) is a newly discovered type of local regulatory factor that mediates its biological effects through the specific, membrane-bound natriuretic peptide receptor-B (NPR-B). Recent studies have established that CNP is closely related to male reproductive function. The aims of this study were to determine the distribution of CNP/NPR-B in human ejaculated spermatozoa through different methods (such as immunolocalization, real time polymerase chain reaction and Western Blot), and then to evaluate the influence of CNP on sperm function in vitro, such as motility and acrosome reaction. Human semen samples were collected from consenting donors who met the criteria of the World Health Organization for normozoospermia. Our results show that the specific receptor NPR-B of CNP is localized in the acrosomal region of the head and the membrane of the front-end tail of the sperm, and there is no signal of CNP in human sperm. Compared with the control, CNP can induce a significant dose-dependent increase in spermatozoa motility and acrosome reaction. In summary, CNP/NPR-B can affect sperm motility and acrosome reaction, thus regulating the reproductive function of males. CNP may be a new key factor in regulating sperm function. PMID:25926602

  3. Molecular mechanisms involved in gamete interaction: evidence for the participation of cysteine-rich secretory proteins (CRISP) in sperm-egg fusion.

    PubMed

    Da Ros, V; Busso, D; Cohen, D J; Maldera, J; Goldweic, N; Cuasnicu, P S

    2007-01-01

    Epididymal protein DE and testicular protein Tpx-1 are two cysteine-rich secretory proteins also known as CRISP-1 and CRISP-2, respectively. DE/ CRISP-1 is localised on the equatorial segment of acrosome-reacted sperm and participates in rat gamete fusion through its binding to egg-complementary sites. Recent results using bacterially-expressed recombinant fragments of DE as well as synthetic peptides revealed that the ability of DE to bind to the egg surface and inhibit gamete fusion resides in a region of 12 amino acids corresponding to an evolutionary conserved motif of the CRISP family (Signature 2). Given the high degree of homology between DE/CRISP-1 and Tpx-1/CRISP-2, we also explored the potential participation of the testicular intra-acrosomal protein in gamete fusion. Results showing the ability of recombinant Tpx-1 to bind to the surface of rat eggs (evaluated by indirect immunofluorescence) and to significantly inhibit zona-free egg penetration, support the participation of this protein in gamete fusion through its interaction with egg-binding sites. Interestingly, rat Tpx-1 exhibits only two substitutions in Signature 2 when compared to this region in DE. Together, these results provide evidence for the involvement of both epididymal DE/CRISP-1 and testicular Tpx-1/CRISP-2 in gamete fusion suggesting the existence of a functional cooperation between homologue molecules as a mechanism to ensure the success of fertilisation.

  4. Ejaculate traits and sperm cryopreservation in the endangered Baird's tapir (Tapirus bairdii).

    PubMed

    Pukazhenthi, Budhan S; Togna, Gina Della; Padilla, Luis; Smith, Diorene; Sanchez, Carlos; Pelican, Katey; Sanjur, Oris I

    2011-01-01

    There is little information on the reproductive biology of the male Baird's tapir (Tapirus bairdii). In this study, we characterized the ejaculate traits and evaluated the efficacy of 2 cryodiluents on sperm cryosurvival. Ejaculates were assessed for volume, pH, sperm motility, forward progression, osmolality, sperm concentration, sperm morphology, and acrosomal integrity. For cryopreservation, ejaculates with >50% total sperm motility were washed, and sperm pellets were resuspended in either Botu-Crio (CryoVital, Grandau, Germany) or INRA 96 containing 2% egg yolk and 2.5% each of methyl- and dimethylformamide (INRA 96), and they were cryopreserved over liquid nitrogen vapor. Thawed samples were incubated in vitro (25 °C) and evaluated for percent total sperm motility, forward progression, and acrosomal integrity at hourly intervals for 4 hours. Spermic ejaculates were obtained from all males, and the mean seminal volume, sperm concentration per milliliter, percent sperm motility, progressive status, and percent morphologically normal cells were 20.4 ± 4.3 mL, 101.2 ± 24.0 × 10(6)/mL, 46.1% ± 5.0%, 2.9 ± 0.1, and 6.9% ± 1.4%, respectively. There was a positive significant correlation between percent normal sperm and animal age (r = 0.66; P < .004). Cryopreservation in either Botu-Crio or INRA 96 resulted in a decline (P < .05) in percent sperm motility and acrosomal integrity. Sperm forward progression remained unaffected immediately after thawing in INRA 96 but continued to decline over time. These results characterize, for the first time, the ejaculate traits of the tapir; demonstrate that tapir spermatozoa can be cryopreserved in diluents containing amides alone or in combination with glycerol; and provide fundamental information critical for development of assisted reproductive technologies for the Baird's tapir.

  5. Spermiogenesis and Taxonomical Values of Sperm Ultrastructures in Male Crassostrea ariakensis (Fujita & Wakiya, 1929) (Pteroirmorphia: Ostreidae) in the Estuary of the Seomjin River, Korea

    PubMed Central

    Son, Pal Won; Chung, Jae Seung; Kim, Jin Hee; Kim, Sung Han; Chung, Ee-Yung

    2014-01-01

    Characteristics of the developmental stages of spermatids during spermiogenesis and phylogenetic classicfication of the species using sperm ultrastructures in male Crassostrea ariakensis were investigated by transmission electron microscope observations. The morphology of the spermatozoon of this species has a primitive type and is similar to those of Ostreidae. Ultrastructures of mature sperms are composed of broad, modified cap-shaped acrosomal vesicle and an axial rod in subacrosomal materials on an oval nucleus, four spherical mitochondria in the sperm midpiece, and satellite fibres which appear near the distal centriole. The axoneme of the sperm tail shows a 9+2 structure. Accordingly, the ultrastructural characteristics of mature sperm of C. ariakensis resemble to those of other investigated ostreids in Ostreidae in the subclass Pteriomorphia. In this study, particularly, two transverse bands (stripes) appear at the anterior region of the acrosomal vesicle of this species, unlike two or three transverse bands (stripes) in C. gigas. It is assumed that differences in this acrosomal substructure are associated with the inability of fertilization between the genus Crassostrea and other genus species in Ostreidae. Therefore, we can use sperm ultrastructures and morphologies in the resolution of taxonomic relationships within the Ostreidae in the subclass Pteriomorphia. These spermatozoa, which contain several ultrastructures such as acrosomal vesicle, an axial rod in the sperm head part and four mitochondria and satellite fibres in the sperm midpiece, belong to the family Ostreidae in the subclass Pteriomorphia. PMID:25949188

  6. [Molecular identification of Ca2+ channels in human ejaculated sperm].

    PubMed

    Li, Lu; Liu, Ji-Hong; Li, Jia-Gui; Ye, Zhang-Qun; Wang, Xiu-Lai

    2006-10-01

    To identify the types of voltage-dependent Ca2+ channels (VDCCs) in human ejaculated sperm using molecular technology. Based on the WHO criteria, the donated semen was screened by computer-aided sperm analysis (CASA), the sperm optimized by swim-up method, and the VDCCs' alpha1 subunits examined by reverse transcription polymerase chain reaction (RT-PCR). Such mRNA messages as alpha1H, alpha1G, alpha1E, alpha1B and alpha1C were detected, but alpha1A and alpha1D were not. Human motile sperms express multiple VDCC RNAs, among which T-type and non-L-type channel messages are likely to be predominantly expressed. Not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, which helps regulate sperm motility and acrosome reaction.

  7. Addition of cholesterol-loaded cyclodextrins to the thawing extender: effects on boar sperm quality.

    PubMed

    Tomás, C; Gómez-Fernández, J; Gómez-Izquierdo, E; Mocé, E; de Mercado, E

    2014-06-01

    The aim of the present study was to evaluate the effect that the addition of cholesterol-loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen-thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg-yolk-based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 10(6) sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p < 0.05) when the thawing extender was supplemented with 12.5, 25 and 50 mg CLC/500 × 10(6) sperm. Nevertheless, motility decreased (p < 0.05) when the concentration of CLC exceeded 12.5 mg CLC/500 × 10(6) sperm. In conclusion, our results suggest that the supplementation of thawing extenders with CLC improves sperm viability and reduces acrosome damage after freezing/thawing.

  8. Sperm DNA integrity in frozen-thawed semen from Italian Mediterranean Buffalo bulls and its relationship to in vivo fertility.

    PubMed

    Serafini, Rosanna; Love, Charles C; Coletta, Angelo; Mari, Gaetano; Mislei, Beatrice; Caso, Chiara; Di Palo, Rossella

    2016-09-01

    The relationship among sperm attributes of DNA integrity, sperm motility, morphology, viability, acrosome integrity and in vivo fertility of frozen-thawed Italian Mediterranean Buffalo (IMB) sperm has not been reported. Straws of frozen-thawed semen samples from three bulls were examined. Sperm DNA assays (i.e., neutral Comet assay, Sperm Bos Halomax-SBH and Sperm Chromatin Structure Assay-SCSA) were not correlated to each other (P>0.05). Many neutral Comet assay measures were correlated to total sperm motility-TMOT (% head-H-DNA, r=0.74; Olive moment, r=-0.76; P<0.05) and coiled tails (r-values ranged from% H-DNA, r=-0.80 to tail length, r=-0.71; P<0.05). The COMP-αt was negatively correlated to viable acrosome intact (VAI) sperm, and distal droplets (r=-0.60 and -0.61; P<0.05), whereas Mean-αt and Mode-αt were positively correlated to bent midpieces (r=0.63 and 0.61; P<0.05). The SBH assay was positively correlated to non-viable acrosome damaged (NVAD) sperm (r=0.60; P<0.05) and negatively correlated to viable acrosome damaged (VAD) sperm (r=-0.63; P<0.05). The overall pregnancy rate (PR-at 30 and 45d post artificial insemination-AI) and the calving rate were 57%, 55% and 45%, respectively. Among sperm features analyzed the area under the Receiver Operating Characteristic (ROC) Curve was significant (P<0.05) for TMOT, NVAD, Standard Deviation-αt (SD-αt) and neutral comet measures (Olive tail moment and tail moment, % H- DNA and tail area) in estimating pregnancy. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Effect of acute/subchronic samarium exposure on the concentration, motility, and morphology of sperm in male mice.

    PubMed

    Zhang, D Y; Shen, X Y; Xu, X L; Ruan, Q; Hu, S S; Chen, Y Y; Wang, Z M

    2016-06-21

    Male ICR mice were orally administered samarium nitrate [Sm(NO3)3] to investigate its effects on sperm concentration and sperm quality. After acute exposure to ≥2880.00 mg/kg Sm(NO3)3 via intragastric gavage, sperm motility and acrosome integrity were decreased, and the sperm malformation percentage was increased (P < 0.05). After subchronic exposure to ≥500.00 mg/L Sm(NO3)3 administered via drinking water for 90 days, relative gonad weight, sperm concentration, and sperm quality significantly decreased (P < 0.05). Sperm malformation also increased after subchronic exposure to Sm, which was found to be the most sensitive index. Sperm head malformation accounted for the largest proportion of all types of sperm malformations evaluated. Of the six different subtypes of head malformation, irregular shape accounted for the largest proportion.

  10. Association between the seminal plasma proteome and sperm functional traits.

    PubMed

    Intasqui, Paula; Camargo, Mariana; Antoniassi, Mariana Pereira; Cedenho, Agnaldo Pereira; Carvalho, Valdemir Melechco; Cardozo, Karina Helena Morais; Zylbersztejn, Daniel Suslik; Bertolla, Ricardo Pimenta

    2016-03-01

    To analyze the seminal plasma proteome and biological functions associated with sperm functional alterations. Cross-sectional study. University andrology and research laboratories. A total of 156 normozoospermic men. Sperm mitochondrial activity, acrosome integrity, and DNA fragmentation were evaluated in a semen aliquot. Remaining semen was centrifuged, and seminal plasma was utilized for proteomic analysis (liquid chromatography-tandem mass spectrometry). Patients were divided into percentiles (15%) to form the following groups: substudy 1, high (control, n = 26) and low (study, n = 23) sperm mitochondrial activity; substudy 2, high (control, n = 23) and low (study, n = 22) sperm acrosome integrity; and substudy 3, low (control, n = 22) and high (study, n = 22) sperm DNA fragmentation. Groups were compared using univariate and multivariate analyses. Differentially expressed proteins were used for functional enrichment analysis. Seminal plasma proteome and postgenomic pathways are associated with several sperm functional traits. In total, 506, 493, and 464 proteins were observed in substudies 1, 2, and 3, respectively. Enriched functions in substudy 1 were intramolecular oxidoreductase activity, aminoglycans catabolism, endopeptidases inhibition, lysosomes, and acute-phase response (study group). In substudy 2, main enriched functions were phospholipase inhibition, arachidonic acid metabolism, exocytosis, regulation of acute inflammation, response to hydrogen peroxide, and lysosomal transport (study group). In substudy 3, enriched functions were prostaglandin biosynthesis and fatty acid binding (study group). We proposed eight, six, and eight seminal biomarkers for substudies 1, 2, and 3, respectively. Seminal plasma proteome reflects sperm mitochondrial activity reduction, acrosome damage, and DNA fragmentation, with several postgenomic functions related to these alterations. Copyright © 2016 American Society for Reproductive Medicine. Published by Elsevier Inc

  11. Rapid freezing without cooling equilibration in canine sperm.

    PubMed

    Kim, Suhee; Lee, Yongcheol; Yang, Honghyun; Kim, Yong-Jun

    2012-01-01

    The aim of this study was to develop a rapid method of canine semen freezing without cooling equilibration using treatment with different cryoprotectant agents (CPAs) and freezing in liquid nitrogen (LN(2)) vapor in a 0.5-mL straw via modifying vitrification. Ejaculates from eight beagle dogs were frozen with different CPAs (CPA-free, 5% glycerol, 5% ethylene glycol, and 10% ethylene glycol) and freezing times (direct plunging into LN(2) or freezing for 1, 2, 3, or 10 min in LN(2) vapor before plunging into LN(2)). Frozen-thawed sperm were evaluated for motility, viability, normal morphology, and plasma- and acrosome-membrane integrities. The 5% glycerol treatment resulted in improved sperm motility, plasma-membrane integrity and acrosome-membrane integrity (P<0.05). Freezing in LN(2) vapor showed improved sperm motility, viability, and plasma membrane integrity (P<0.05), and freezing for more than 2 min in LN(2) vapor increased acrosome-membrane integrity compared with direct plunging into LN(2) (P<0.05). The direct plunging into LN(2) showed no motile sperm. However, freezing for more than 2 min in LN(2) vapor increased the total abnormalities compared to direct plunging into LN(2) (P<0.05). In conclusion, use of 5% glycerol and freezing in LN(2) vapor were essential for the rapid freezing of canine sperm without cooling equilibration. In particular, holding for 2 min in LN(2) vapor was sufficient to yield successful rapid freezing. This rapid freezing method is simple and effective in canine sperm and would be helpful to offer information for trial of vitrification in large volumes of canine sperm.

  12. Use of Androcoll-S after thawing improves the quality of electroejaculated and epididymal sperm samples from red deer.

    PubMed

    Anel-López, L; Martínez-Rodríguez, C; Soler, A J; Fernández-Santos, M R; Garde, J J; Morrell, J M

    2015-07-01

    Single Layer Centrifugation is a useful technique to select sperm with good quality. The use of selection methods such as Androcoll could become an important tool to improve the quality of sperm samples and therefore to improve other artificial reproductive techniques such as sperm sex sorting, in vitro fertilization or AI. The aim of this study was to evaluate the effect of a Single Layer Centrifugation with Androcoll-S on the sperm quality of red deer sperm samples of two different origins, electroejaculated samples and epididymal samples obtained post-mortem, after thawing and after an incubation for 2h at 37°C. Sperm motility, viability, membrane permeability, mitochondrial activity, acrosomal status and DNA fragmentation were determined for all samples. The samples selected by Androcoll-S showed an improvement in sperm kinematics compared to unselected samples after thawing and after incubation. The same effect was observed in parameters such as viability, mitochondrial activity or acrosomal status which were improved after the selection. In contrast, no difference was found in DNA fragmentation between selected and unselected samples within the same sperm type. We conclude that sperm selection by SLC with Androcoll-S after thawing for red deer sperm of both types is a suitable technique that allows sperm quality in both types of sperm samples to be improved, thereby improving other assisted reproductive techniques. Further studies (IVF and in vivo fertilization) are required to determine whether this improvement can increase fertility, as has been shown for other species.

  13. Partial Deletion of Chromosome 8 β-defensin Cluster Confers Sperm Dysfunction and Infertility in Male Mice

    PubMed Central

    Zhou, Yu S.; Webb, Sheila; Lettice, Laura; Tardif, Steve; Kilanowski, Fiona; Tyrrell, Christine; MacPherson, Heather; Semple, Fiona; Tennant, Peter; Baker, Tina; Hart, Alan; Devenney, Paul; Perry, Paul; Davey, Tracey; Barran, Perdita; Barratt, Chris L.; Dorin, Julia R.

    2013-01-01

    β-defensin peptides are a family of antimicrobial peptides present at mucosal surfaces, with the main site of expression under normal conditions in the male reproductive tract. Although they kill microbes in vitro and interact with immune cells, the precise role of these genes in vivo remains uncertain. We show here that homozygous deletion of a cluster of nine β-defensin genes (DefbΔ9) in the mouse results in male sterility. The sperm derived from the mutants have reduced motility and increased fragility. Epididymal sperm isolated from the cauda should require capacitation to induce the acrosome reaction but sperm from the mutants demonstrate precocious capacitation and increased spontaneous acrosome reaction compared to wild-types but have reduced ability to bind the zona pellucida of oocytes. Ultrastructural examination reveals a defect in microtubule structure of the axoneme with increased disintegration in mutant derived sperm present in the epididymis cauda region, but not in caput region or testes. Consistent with premature acrosome reaction, sperm from mutant animals have significantly increased intracellular calcium content. Thus we demonstrate in vivo that β-defensins are essential for successful sperm maturation, and their disruption leads to alteration in intracellular calcium, inappropriate spontaneous acrosome reaction and profound male infertility. PMID:24204287

  14. Ketamine inhibits human sperm function by Ca(2+)-related mechanism.

    PubMed

    He, Yuanqiao; Zou, Qianxing; Li, Bingda; Chen, Houyang; Du, Xiaohong; Weng, Shiqi; Luo, Tao; Zeng, Xuhui

    2016-09-09

    Ketamine, a dissociative anesthetic, which was widely used in human and animal medicine, has become a popular recreational drug, as it can induce hallucinatory effects. Ketamine abuse can cause serious damage to many aspects of the organism, mainly reflected in the nervous system and urinary system. It has also been reported that ketamine can impair the male genital system. However, the detailed effect of ketamine on human spermatozoa remains unclear. Thus, we investigated the in vitro effects of ketamine on human sperm functions, to elucidate the underlying mechanism. Human sperm were treated in vitro with different concentrations of ketamine (0, 0.125, 0.25, 0.5, 1 g/L). The results showed that 0.25-1 g/L ketamine inhibited sperm total motility, progressive motility and linear velocity, in a dose-dependent manner. In addition, the sperm's ability to penetrate viscous medium and the progesterone-induced acrosome reaction were significantly inhibited by ketamine. Ketamine did not affect sperm viability, capacitation and spontaneous acrosome reaction. The intracellular calcium concentration ([Ca(2+)]i), which is a central factor in the regulation of human sperm function, was decreased by ketamine (0.125-1 g/L) in a dose-dependent manner. Furthermore, the currents of the sperm-specific Ca(2+) channel, CatSper, which modulates Ca(2+) influx in sperm, were inhibited by ketamine (0.125-1 g/L) in a dose-dependent manner. Our findings suggest that ketamine induces its toxic effects on human sperm functions by reducing sperm [Ca(2+)]i through inhibition of CatSper channel. Copyright © 2016 Elsevier Inc. All rights reserved.

  15. Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: Light and electron microscopy

    USGS Publications Warehouse

    Walker, G.K.; Black, M.G.; Edwards, C.A.

    1996-01-01

    Adult zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondrial "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria.

  16. Cryopreservation of human spermatozoa. I. Effects of holding procedure and seeding on motility, fertilizability, and acrosome reaction.

    PubMed

    Critser, J K; Huse-Benda, A R; Aaker, D V; Arneson, B W; Ball, G D

    1987-04-01

    Three experiments were conducted to evaluate effects of holding semen at +5.0 degrees C for 30 minutes or -5.0 degrees C for 10 minutes and ice crystal induction (seeding) on frozen-thawed human spermatozoa. In experiment 1, spermatozoa were frozen, and postthaw motility was evaluated immediately (0 hour) and 24 hours later. At both 0 and 24 hours, nonfrozen control samples had higher motility than all other treatment groups. At 0 hour postthaw, motility was higher in samples held at -5.0 degrees C for 10 minutes with no significant effect of seeding. At 24 hours, samples held at -5.0 degrees C for 10 minutes and seeded, but not samples held at -5.0 degrees C and not seeded, had higher motility than samples held at +5.0 degrees C. In experiment 2, semen samples were frozen, and fertilizability was evaluated in a zona-free hamster egg penetration assay. Seeded samples had a higher frequency of sperm penetration than either nonfrozen or nonseeded samples. In experiment 3, nonfrozen controls and frozen treatment groups were evaluated for the frequency of survival and acrosomal integrity. Seeded samples had higher frequencies of survival and loss of acrosomal integrity than nonseeded samples. All frozen-thawed samples had a lower frequency of survival and a higher frequency of loss of acrosomal integrity than nonfrozen controls. Although altered patterns of fertilizability and acrosomal integrity are induced, collectively these data suggest that incorporating a holding temperature of -5.0 degrees C for 10 minutes and seeding may result in a superior protocol for freezing human spermatozoa.

  17. In vitro effects of nonylphenol on motility, mitochondrial, acrosomal and chromatin integrity of ram and boar spermatozoa.

    PubMed

    Uguz, C; Varisli, O; Agca, C; Evans, T; Agca, Y

    2015-10-01

    The objective of this study was to determine the effects of nonylphenol (NP) on viability of ram and boar sperm in vitro. Ram or boar spermatozoa were exposed to 1, 10, 100, 250 and 500 μg NP ml(-1) for 1, 2, 3 or 4 h. Computer-assisted sperm motility analysis (CASA) system was used to evaluate sperm motility characteristics. Flow cytometry was used to determine mitochondrial membrane potential (MMP) and chromatin integrity, while epifluorescent microscopy was used to determine sperm acrosomal status. Exposure of both species spermatozoa to 250 and 500 μg NP ml(-1) was detrimental to progressive motility (P < 0.05), and its adverse effect was significant at lower (100 μg NP ml(-1) ) concentration (P < 0.05). The percentages of ram and boar spermatozoa with high MMP declined drastically after exposures to ≥250 μg ml(-1) NP (P < 0.05). Unlike chromatin integrity, which did not appear to be altered by NP exposure, there were dose-dependent NP effects (P < 0.05) on acrosomal integrity of both species at as low as 1 μg ml(-1) NP for boar spermatozoa and 10 μg ml(-1) NP for ram spermatozoa. These data show adverse effects of NP on ram and boar spermatozoa and thus its potential harmful effects on male reproduction as NP is found in fruits, vegetables, human milk, fish and livestock products.

  18. Human spermatozoa possess a calcium-dependent chloride channel that may participate in the acrosomal reaction

    PubMed Central

    Orta, Gerardo; Ferreira, Gonzalo; José, Omar; Treviño, Claudia L; Beltrán, Carmen; Darszon, Alberto

    2012-01-01

    Motility, maturation and the acrosome reaction (AR) are fundamental functions of mammalian spermatozoa. While travelling through the female reproductive tract, spermatozoa must mature through a process named capacitation, so that they can reach the egg and undergo the AR, an exocytotic event necessary to fertilize the egg. Though Cl− is important for sperm capacitation and for the AR, not much is known about the molecular identity of the Cl− transporters involved in these processes. We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa. Our whole cell recordings revealed the presence of a Ca2+-dependent Cl− current. The biophysical characteristics of this current and its sensitivity to niflumic acid (NFA) and 4,4′-diisothiocyano-2,2′-stilbene disulphonic acid (DIDIS) are consistent with those displayed by the Ca2+-dependent Cl− channel from the anoctamin family (TMEM16). Whole cell patch clamp recordings in the cytoplasmic droplet of human spermatozoa corroborated the presence of these currents, which were sensitive to NFA and to a small molecule TMEM16A inhibitor (TMEM16Ainh, an aminophenylthiazole). Importantly, the human sperm AR induced by a recombinant human glycoprotein from the zona pellucida, rhZP3, displayed a similar sensitivity to NFA, DIDS and TMEM16Ainh as the sperm Ca2+-dependent Cl− currents. Our findings indicate the presence of Ca2+-dependent Cl− currents in human spermatozoa, that TMEM16A may contribute to these currents and also that sperm Ca2+-dependent Cl− currents may participate in the rhZP3-induced AR. PMID:22473777

  19. Not All Sperm Are Equal: Functional Mitochondria Characterize a Subpopulation of Human Sperm with Better Fertilization Potential

    PubMed Central

    Sousa, Ana Paula; Amaral, Alexandra; Baptista, Marta; Tavares, Renata; Caballero Campo, Pedro; Caballero Peregrín, Pedro; Freitas, Albertina; Paiva, Artur; Almeida-Santos, Teresa; Ramalho-Santos, João

    2011-01-01

    Human sperm samples are very heterogeneous and include a low amount of truly functional gametes. Distinct strategies have been developed to characterize and isolate this specific subpopulation. In this study we have used fluorescence microscopy and fluorescence-activated cell sorting to determine if mitochondrial function, as assessed using mitochondrial-sensitive probes, could be employed as a criterion to obtain more functional sperm from a given ejaculate. We first determined that mitochondrial activity correlated with the quality of distinct human samples, from healthy donors to patients with decreased semen quality. Furthermore, using fluorescence-activated cell sorting to separate sperm with active and inactive mitochondria we found that this was also true within samples. Indeed, sperm with active mitochondria defined a more functional subpopulation, which contained more capacitated and acrosome intact cells, sperm with lower chromatin damage, and, crucially, sperm more able to decondense and participate in early development using both chemical induction and injection into mature bovine oocytes. Furthermore, cell sorting using mitochondrial activity produced a more functional sperm subpopulation than classic swim-up, both in terms of improvement in a variety of functional sperm parameters and in statistical significance. In conclusion, whatever the true biological role of sperm mitochondria in fertilization, mitochondrial activity is a clear hallmark of human sperm functionality. PMID:21448461

  20. Evaluation of Lasting Effects of Heat Stress on Sperm Profile and Oxidative Status of Ram Semen and Epididymal Sperm

    PubMed Central

    Hamilton, Thais Rose dos Santos; Mendes, Camilla Mota; de Castro, Letícia Signori; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Delgado, Juliana de Carvalho; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; Assumpção, Mayra Elena Ortiz D'Ávila

    2016-01-01

    Higher temperatures lead to an increase of testicular metabolism that results in spermatic damage. Oxidative stress is the main factor responsible for testicular damage caused by heat stress. The aim of this study was to evaluate lasting effects of heat stress on ejaculated sperm and immediate or long-term effects of heat stress on epididymal sperm. We observed decrease in motility and mass motility of ejaculated sperm, as well as an increase in the percentages of sperm showing major and minor defects, damaged plasma and acrosome membranes, and a decrease in the percentage of sperm with high mitochondrial membrane potential in the treated group until one spermatic cycle. An increased enzymatic activity of glutathione peroxidase and an increase of stressed cells were observed in ejaculated sperm of the treated group. A decrease in the percentage of epididymal sperm with high mitochondrial membrane potential was observed in the treated group. However, when comparing immediate and long-term effects, we observed an increase in the percentage of sperm with low mitochondrial membrane potential. In conclusion, testicular heat stress induced oxidative stress that led to rescuable alterations after one spermatic cycle in ejaculated sperm and also after 30 days in epididymal sperm. PMID:26881013

  1. Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls.

    PubMed

    Reis, L S L S; Ramos, A A; Camargos, A S; Oba, E

    2014-11-10

    The effect of dietary manganese (Mn(2+)) supplementation on the reproductive performance of Nelore bulls was evaluated by assessment of sperm membrane integrity. Sixty Nelore bulls (Bos taurus indicus) aged 18-20 mo were randomly divided into four groups (n=15) receiving dietary Mn(2+) supplementation at 540, 1300, 3800 and 6300mg/kg (treatments TC, T1300, T3800 and T6300, respectively). The diets were changed for the groups every 70d. Semen samples were obtained 15 and 56d after the diet change, which corresponded to the period of adjustment to the new diet and the time required for a complete spermatogenesis cycle, respectively. Sperm integrity was assessed by detection of: intact (IMe) or damaged (DMe) membranes, intact (IA) or damaged (DA) acrosomes, and high (HM) or low (LM) mitochondrial membrane potentials. Only bulls from the TC treatment showed a significant increase in the production of intact sperm [IMe/IA/LM] and decrease in the production of sperm with damaged acrosome [IMe/DA/LM] or completely damaged sperm [DMe/DA/LM] (P<0.05). The Mn(2+) concentrations in the semen were positively correlated with the incidence of sperm with IMe, DA, and LM and negatively correlated with number of sperm with DMe, IA, and LM. Therefore, dietary Mn(2+) supplementation for Nelore bulls must be limited to 540mg of Mn(2+)/kg given that higher doses are detrimental to the integrity of the plasma and acrosomal sperm membranes.

  2. Human sperm quality and lipid content after migration into normal ovulatory human cervical mucus containing low numbers of leukocytes

    PubMed Central

    Chakroun-Feki, Nozha; Therond, Patrice; Couturier, Martine; Eustache, Florence; Limea, Gerard; Legrand, Alain; Jouannet, Pierre; Auger, Jacques

    2009-01-01

    The aim of this study was to investigate whether a relationship exists between the presence of low numbers of leukocytes in normal ovulatory cervical mucus and sperm quality and lipid content after migration. The percentages of live, motile and morphologically normal spermatozoa, movement parameters assessed by computer-aided sperm analysis (CASA), and ionophore-induced acrosome reaction measured by flow cytometry were determined before and after migration. High-performance liquid chromatography with ultraviolet detection was used to measure the sperm lipid content, including the various diacyl subspecies. The number of leukocytes found in solubilized mucus samples was counted using a haemocytometric method. Overall, the presence of leukocytes in the cervical mucus samples did not significantly influence sperm motility and morphology, sperm kinematic parameters, or the sperm content in sphingomyelin or cholesterol. In contrast, after migration, the decrease in various sperm diacyls and the level of induced acrosome reaction was significantly less pronounced in mucus samples containing ≥ 104 leukocytes than in mucus samples with no or rare leukocytes whereas the level of induced acrosome reaction was higher. The present data suggest that the low level of leukocytes found in normal ovulatory cervical mucus could influence the process of sperm lipid remodelling/capacitation. PMID:19182821

  3. Speract, a sea urchin egg peptide that regulates sperm motility, also stimulates sperm mitochondrial metabolism.

    PubMed

    García-Rincón, Juan; Darszon, Alberto; Beltrán, Carmen

    2016-04-01

    Sea urchin sperm have only one mitochondrion, that in addition to being the main source of energy, may modulate intracellular Ca(2+) concentration ([Ca(2+)]i) to regulate their motility and possibly the acrosome reaction. Speract is a decapeptide from the outer jelly layer of the Strongylocentrotus purpuratus egg that upon binding to its receptor in the sperm, stimulates sperm motility, respiration and ion fluxes, among other physiological events. Altering the sea urchin sperm mitochondrial function with specific inhibitors of this organelle, increases [Ca(2+)]i in an external Ca(2+) concentration ([Ca(2+)]ext)-dependent manner (Ardón, et al., 2009. BBActa 1787: 15), suggesting that the mitochondrion is involved in sperm [Ca(2+)]i homeostasis. To further understand the interrelationship between the mitochondrion and the speract responses, we measured mitochondrial membrane potential (ΔΨ) and NADH levels. We found that the stimulation of sperm with speract depolarizes the mitochondrion and increases the levels of NADH. Surprisingly, these responses are independent of external Ca(2+) and are due to the increase in intracellular pH (pHi) induced by speract. Our findings indicate that speract, by regulating pHi, in addition to [Ca(2+)]i, may finely modulate mitochondrial metabolism to control motility and ensure that sperm reach the egg and fertilize it.

  4. Quantitative changes of Ricinus communis agglutinin I and Helix pomatia lectin binding sites in the acrosome of rat spermatozoa during epididymal transit.

    PubMed

    Hermo, L; Winikoff, R; Kan, F W

    1992-09-01

    During passage through the epididymis, spermatozoa undergo a number of changes which result in their acquisition of fertility and motility. Some of the changes that occur include loss of the cytoplasmic droplet and changes in sperm morphology, metabolism and properties of the nucleus and plasma membrane. Changes have also been reported in the acrosomic system of mammalian spermatozoa during their transit through the epididymis. In the present study, the quantitative changes of the glycoconjugate content in the acrosome of rat spermatozoa were examined during their passage through the epididymis using lectin-colloidal gold cytochemistry. Various regions of the epididymis (initial segment, caput, corpus and cauda epididymidis) were fixed by perfusion with 1% or 2% glutaraldehyde buffered in sodium cacodylate (0.1 M), dehydrated in ethanol and embedded without osmication in Lowicryl K4M. Lectin-colloidal gold labeling was performed on thin sections using Ricinus communis agglutinin I (RCA I) or Helix pomatia lectin (HPL) to detect D-galactose- and N-acetyl-D-galactosamine-containing glycoconjugates, respectively. The labeling density over the acrosome of the acrosomic system was evaluated as the number of gold particles per microns 2 of profile area using a Zeiss MOP-3 image analyzer. The overall mean labeling densities over the acrosome of spermatozoa for each lectin was estimated from 4 rats and over the four distinct epididymal regions. The mean labeling density of the acrosome with RCA I and HPL showed a similar pattern along the epididymis, although RCA I revealed approximately twice as many gold particles per epididymal region. In either case, there was a significant decrease in the labeling density of the acrosome of spermatozoa between the initial segment or caput epididymidis and cauda epididymidis (p less than 0.01). A similar decrease was also noted between the initial segment and corpus epididymidis (p less than 0.01). No change was found between the

  5. Insights into the lysine acetylproteome of human sperm.

    PubMed

    Sun, Guohai; Jiang, Min; Zhou, Tao; Guo, Yueshuai; Cui, Yiqiang; Guo, Xuejiang; Sha, Jiahao

    2014-09-23

    Protein lysine acetylation is a dynamic and reversible post-modification that is known to play diverse functions in eukaryotes. Nevertheless, the composition and function of non-histone lysine acetylation in gametes remain unknown. In humans, only capacitated sperm have the capacity to fertilize an egg. In the present study, we found complex composition of lysine acetylated proteins in capacitated human sperm. In vitro fertilization inhibition assay by anti-acetyllysine antibody showed essential roles of lysine acetylation in fertilization. And inhibition of lysine deacetylases, the histone deacetylases, by trichostatin A and nicotinamide, could significantly suppress sperm motility. After immunopurification enrichment of acetylpeptides with anti-acetyllysine antibody and high-throughput liquid chromatography-tandem mass spectrometry identification, we characterized 1206 lysine acetylated sites, corresponding to 576 lysine acetylated proteins in human capacitated sperm. Bioinformatics analysis showed that these proteins are associated with sperm functions, including motility, capacitation, acrosome reaction and sperm-egg interaction. Thus, lysine acetylation is expected to be an important regulatory mechanism for sperm functions. And our characterization of lysine acetylproteome could be a rich resource for the study of male fertility. Mature sperm are almost transcriptionally and translationally silent, thus post-translational modifications play important roles in sperm functions. Till now, only two types of PTMs, phosphorylation and glycosylation, are well studied in normal human sperm based on large scale proteomics. In the present study, we established the acetylproteome of capacitated human sperm. Over 1000 lysine acetylated sites were identified. Bioinformatics analysis shows that lysine acetylated proteins participate in many biological events of sperm functions. We further provided functional data that the lysine acetylation is essential for sperm motility

  6. Sperm Bundles in the Seminal Vesicle of the Crematogaster victima (Smith) Adult Males (Hymenoptera: Formicidae).

    PubMed

    Oliveira, C M; Moreira, J; Gomes, L F; Camargo-Mathias, M I; Lino-Neto, J

    2014-06-01

    This study establishes the presence of spermatodesm in the seminal vesicles of sexually mature males of Crematogaster victima (Smith). In this species, the spermatozoa are maintained together by an extracellular matrix in which the acrosomal regions are embedded. This characteristic has not yet been observed in any other Aculeata. However, the sperm morphology in this species is similar to that described for other ants. The spermatozoa measure on average 100 μm in length, and the number of sperm per bundle is up to 256. They are composed of a head formed by the acrosome and nucleus; this is followed by the flagellum, which is formed by the centriolar adjunct, an axoneme with a 9 + 9 + 2 microtubule pattern, two mitochondrial derivatives, and two accessory bodies. The acrosome is formed by the acrosomal vesicle and perforatorium. The nucleus is filled with compact chromatin with many areas of thick and non-compacted filaments. Both mitochondrial derivatives have the same shape and diameters. The presence of sperm bundles in sexually mature males differentiates C. victima from other ants; however, the similarities in the sperm ultrastructure support the monophyly of this insect group.

  7. Presence of F-actin in sperm head of Armadillidium peraccae (Isopoda, Oniscidea).

    PubMed

    Trovato, M; Mazzei, V; Sinatra, F; Longo, G

    2011-10-01

    Sperm of Armadillidium peraccae have been examined with cytochemical and immunocytochemical methods for fluorescence and electron microscopic visualization of cytoskeleton components. Sperm incubation in an antibody anti-β-tubulin shows only the presence of two centrioles located in the cytoplasmic region above the nucleus; no other microtubules are present in the sperm head. Instead, fluorescence microscopy of sperm incubated in FITC-phalloidin allowed to detect the presence of a large amount of F-actin in the apical region of the sperm head. The incubation of ultrathin sections of sperm embedded in Lowicryl K4M with a phalloidin-gold complex allowed a more precise localization of F-actin in the amorphous part of the acrosome and in the cytoplasmic region between acrosome and nucleus; F-actin is also present in the thin cytoplasmic layer between plasma membrane and nuclear envelope at the apical portion of the nucleus. Although the sperm was always found completely devoid of motility, the discovery of the presence of an actin cytoskeleton leads us to hypothesize a possible acquisition of motility by the sperm at the time of its interaction with the female gamete. Such a hypothesis is supported by what is known for ostracods whose aflagellate sperm implement a type of amoeboid movement only at the time of their interaction with the female gamete.

  8. Chemical reacting flows

    NASA Astrophysics Data System (ADS)

    Mularz, Edward J.; Sockol, Peter M.

    1990-02-01

    Future aerospace propulsion concepts involve the combustion of liquid or gaseous fuels in a highly turbulent internal airstream. Accurate predictive computer codes which can simulate the fluid mechanics, chemistry, and turbulence-combustion interaction of these chemical reacting flows will be a new tool that is needed in the design of these future propulsion concepts. Experimental and code development research is being performed at LeRC to better understand chemical reacting flows with the long-term goal of establishing these reliable computer codes. Our approach to understand chemical reacting flows is to look at separate, more simple parts of this complex phenomenon as well as to study the full turbulent reacting flow process. As a result, we are engaged in research on the fluid mechanics associated with chemical reacting flows. We are also studying the chemistry of fuel-air combustion. Finally, we are investigating the phenomenon of turbulence-combustion interaction. Research, both experimental and analytical, is highlighted in each of these three major areas.

  9. Chemical reacting flows

    NASA Technical Reports Server (NTRS)

    Mularz, Edward J.; Sockol, Peter M.

    1990-01-01

    Future aerospace propulsion concepts involve the combustion of liquid or gaseous fuels in a highly turbulent internal airstream. Accurate predictive computer codes which can simulate the fluid mechanics, chemistry, and turbulence-combustion interaction of these chemical reacting flows will be a new tool that is needed in the design of these future propulsion concepts. Experimental and code development research is being performed at LeRC to better understand chemical reacting flows with the long-term goal of establishing these reliable computer codes. Our approach to understand chemical reacting flows is to look at separate, more simple parts of this complex phenomenon as well as to study the full turbulent reacting flow process. As a result, we are engaged in research on the fluid mechanics associated with chemical reacting flows. We are also studying the chemistry of fuel-air combustion. Finally, we are investigating the phenomenon of turbulence-combustion interaction. Research, both experimental and analytical, is highlighted in each of these three major areas.

  10. Chemical reacting flows

    NASA Technical Reports Server (NTRS)

    Mularz, Edward J.; Sockol, Peter M.

    1987-01-01

    Future aerospace propulsion concepts involve the combination of liquid or gaseous fuels in a highly turbulent internal air stream. Accurate predictive computer codes which can simulate the fluid mechanics, chemistry, and turbulence combustion interaction of these chemical reacting flows will be a new tool that is needed in the design of these future propulsion concepts. Experimental and code development research is being performed at Lewis to better understand chemical reacting flows with the long term goal of establishing these reliable computer codes. The approach to understanding chemical reacting flows is to look at separate simple parts of this complex phenomena as well as to study the full turbulent reacting flow process. As a result research on the fluid mechanics associated with chemical reacting flows was initiated. The chemistry of fuel-air combustion is also being studied. Finally, the phenomena of turbulence-combustion interaction is being investigated. This presentation will highlight research, both experimental and analytical, in each of these three major areas.

  11. Effect of sod (superoxide dismutase) protein supplementation in semen extenders on motility, viability, acrosome status and ERK (extracellular signal-regulated kinase) protein phosphorylation of chilled stallion spermatozoa.

    PubMed

    Cocchia, N; Pasolini, M P; Mancini, R; Petrazzuolo, O; Cristofaro, I; Rosapane, I; Sica, A; Tortora, G; Lorizio, R; Paraggio, G; Mancini, A

    2011-04-15

    New studies are underway to find new methods for supporting longer storage of cooled stallion semen. It is known that high concentrations of reactive oxygen species (ROS) cause sperm pathology. The metalloprotein superoxide dismutase (SOD) is responsible for H(2)O(2) and O(2) production, by dismutation of superoxide radicals. The aim of this study is to assess the quality of chilled stallion semen processed with extenders containing SOD at different concentrations as antioxidant additives. A total of 80 ejaculates collected from 5 standardbred stallions was divided into 5 aliquots treated as: native semen (control 1); native semen diluted 1:3 with Kenney semen extender (control 2); spermatozoa diluted after centrifugation in extender without (control 3) or with SOD at 25 IU/ml (experimental 1) or 50 IU/ml (experimental 2). Each sample was analyzed for motility, viability and acrosome status, immediately after semen preparation and again after storage at 5 °C for 24 h, 48 h and 7 2h. Acrosome integrity was evaluated by Chlortetracycline (CTC) and Fluorescent-labeled peanut lectin agglutinin (PNA-FITC conjugated staining). A proteomic approach of quantifying extracellular signal regulated kinase (ERK) was also evaluated as an indirect indicator of oxidative stress. In all samples sperm progressive motility and sperm acrosomal integrity showed a significant reduction between fresh and cooled spermatozoa at 24 h, 48 h and 72 h. Quality parameters of sperm were significantly higher (Progressive Motility P < 0.01; Viability P < 0.001) in aliquots supplemented with SOD. ERK phosphorylation was statistically higher (P < 0.01) in aliquots without SOD. The Authors concluded that addition of SOD to semen extenders improves the quality of chilled equine semen and reduces ERK activation.

  12. Supersonic reacting internal flowfields

    NASA Astrophysics Data System (ADS)

    Drummond, J. P.

    The national program to develop a trans-atmospheric vehicle has kindled a renewed interest in the modeling of supersonic reacting flows. A supersonic combustion ramjet, or scramjet, has been proposed to provide the propulsion system for this vehicle. The development of computational techniques for modeling supersonic reacting flowfields, and the application of these techniques to an increasingly difficult set of combustion problems are studied. Since the scramjet problem has been largely responsible for motivating this computational work, a brief history is given of hypersonic vehicles and their propulsion systems. A discussion is also given of some early modeling efforts applied to high speed reacting flows. Current activities to develop accurate and efficient algorithms and improved physical models for modeling supersonic combustion is then discussed. Some new problems where computer codes based on these algorithms and models are being applied are described.

  13. Ion channels: Key elements in sea urchin sperm physiology

    NASA Astrophysics Data System (ADS)

    Darszon, Alberto; de De Latorre, Lucia; Vargas, Irma; Liévano, Arturo; Beltrán, Carmen; Santi, Celia; Labarca, Pedro; Zapata, Otilia

    1995-08-01

    Ion channels are deeply involved in sea urchin sperm activation, motility, chemotaxis and in the acrosome reaction. Unraveling ion channel function and regulation in sperm behavior has required a combination of complementary approaches since spermatozoa are very tiny cells. Planar bilayer and patch clamp techniques have allowed us to detect, for the first time, the activity of single channels in the plasma membrane of these cells. Unlike intact sperm, swollen sperm can be much more easily patch clamped and single channel activity recorded. These techniques, together with studies of membrane potential, intracellular Ca2+ and pH in whole sperm, have established the presence of K+, Ca2+, and Cl- channels in this cell. The strategies developed to study sea urchin sperm channels are applicable to mammalian spermatozoa. We recently detected a Ca2+ channel resembling one found in S. purpuratus sperm in planar bilayers containing mouse sperm plasma membranes. The presence of this Ca2+ channel in such diverse species suggests it is important in sperm function.

  14. Osteopontin improves sperm capacitation and in vitro fertilization efficiency in buffalo (Bubalus bubalis).

    PubMed

    Boccia, Lucia; Di Francesco, Serena; Neglia, Gianluca; De Blasi, Marina; Longobardi, Valentina; Campanile, Giuseppe; Gasparrini, Bianca

    2013-08-01

    The aim of this study was to evaluate the effect of osteopontin (OPN), an ubiquitous acid glycoprotein, on in vitro sperm capacitation and on in vitro embryo production (IVEP) efficiency in buffalo. In experiment 1, after swim-up separation the sperm were incubated in Tyrode albumin lactate pyruvate medium in the absence of capacitating agents (control), with the standard concentration of heparin (0.01 mM) and three different concentrations of OPN (0.1, 1, and 10 mcg/mL), both in the presence and absence of heparin, for 2 and 4 hours. Capacitation was assessed indirectly by estimating the percentage of acrosome-reacted sperm after incubation with lysophosphatidylcholine. In order to determine the effect of OPN, in the presence of heparin, on fertilization (Experiment 2) and in vitro embryo development (experiment 3), in vitro-matured buffalo oocytes were fertilized in the presence of 0, 0.1, 1, and 10 mcg/mL of OPN. After IVF, the presumptive zygotes were dezonated, fixed, stained, and then evaluated microscopically. At Days 5 and 7 of culture, the cleavage and blastocyst rates were evaluated, respectively. Two hours of treatment with OPN at the two higher concentrations (1 and 10 mcg/mL) promoted in vitro capacitation of buffalo sperm (experiment 1). A synergic action of OPN with heparin was also done for all OPN concentrations tested. At 4 hours incubation, all treatments, including heparin (20.4%), improved (P < 0.01) capacitation compared with the control (16.2%). Interestingly, the best results were reported in all groups treated with OPN + heparin (40.8%, 38.6%, and 33.8%, respectively; P < 0.01). The addition of OPN to the IVF medium had a positive influence on total penetration, synchronous pronuclei formation (experiment 2), and IVEP efficiency (experiment 3). In particular, the two lower concentrations of OPN (0.1 and 1 mcg/mL), compared with the control, gave higher synchronous pronuclei formation (73.5%, 75.0%, and 46.5%, respectively; P < 0.01) and

  15. Two-dimensional polyacrylamide gel electrophoresis of membrane proteins from flow cytometrically sorted ram sperm.

    PubMed

    Leahy, T; Marti, J I; Crossett, B; Evan, G; Maxwell, W M C

    2011-03-15

    Membrane proteins orchestrate key events required for participation of sperm in fertilisation. These proteins may be removed or altered due to the mechanical and dilution stressors associated with sex-sorting of sperm. Ram sperm were incubated with Hoechst 33342 and flow-sorted. Sex-selected (viable, orientated) and waste (separated into non-viable or non-orientated) sperm populations were collected, or sperm were not sorted. Sperm membrane proteins were extracted and characterised by one- and two-dimensional PAGE. Densiometric analysis of protein bands separated by one-dimensional PAGE showed proteins of 30 and 28 kDa as doublet bands on non-sorted sperm, and single bands on sex-sorted sperm, and the proportion of a 14 kDa protein was 3-fold higher in non-sorted compared to sorted sperm. Proteins in the 14 kDa band were identified by mass spectroscopy as a bovine Fibronectin type-2 protein (Fn-2), cytochrome oxidase 5a (Cox5a) and a sperm membrane associated protein (SLLP1). The abundance of these proteins in the two-dimensional gels was lowest in the sorted sperm population identified as viable during sorting (orientated and non-orientated sperm) and highest in the non-viable sperm population (P < 0.001). We concluded that the membrane protein profile was different for sex-sorted compared with non-sorted sperm, due to the selection of plasma membrane-intact cells in the flow-sorted population. This provided further evidence that sex-sorting selected a homogenous population of sperm with superior function to non-sorted sperm. Furthermore, this was apparently the first time sperm membrane acrosome associated protein was reported in ram sperm, and it was demonstrated that seminal plasma proteins remained on the sperm membrane after sex-sorting. Copyright © 2011 Elsevier Inc. All rights reserved.

  16. Rediscovering sperm ion channels with the patch-clamp technique

    PubMed Central

    Kirichok, Yuriy; Lishko, Polina V.

    2011-01-01

    Upon ejaculation, mammalian spermatozoa have to undergo a sequence of physiological transformations within the female reproductive tract that will allow them to reach and fertilize the egg. These include initiation of motility, hyperactivation of motility and perhaps chemotaxis toward the egg, and culminate in the acrosome reaction that permits sperm to penetrate the protective vestments of the egg. These physiological responses are triggered through the activation of sperm ion channels that cause elevations of sperm intracellular pH and Ca2+ in response to certain cues within the female reproductive tract. Despite their key role in sperm physiology and their absolute requirement for the process of fertilization, sperm ion channels remain poorly understood due to the extreme difficulty in application of the patch-clamp technique to spermatozoa. This review covers the topic of sperm ion channels in the following order: first, we discuss how the intracellular Ca2+ and pH signaling mediated by sperm ion channels controls sperm behavior during the process of fertilization. Then, we briefly cover the history of the methodology to study sperm ion channels, which culminated in the recent development of a reproducible whole-cell patch-clamp technique for mouse and human cells. We further discuss the main approaches used to patch-clamp mature mouse and human spermatozoa. Finally, we focus on the newly discovered sperm ion channels CatSper, KSper (Slo3) and HSper (Hv1), identified by the sperm patch-clamp technique. We conclude that the patch-clamp technique has markedly improved and shifted our understanding of the sperm ion channels, in addition to revealing significant species-specific differences in these channels. This method is critical for identification of the molecular mechanisms that control sperm behavior within the female reproductive tract and make fertilization possible. PMID:21642646

  17. Acrosin activity is a good predictor of boar sperm freezability.

    PubMed

    Pinart, Elisabeth; Yeste, Marc; Bonet, Sergi

    2015-06-01

    The main aim of this study was to determine whether acrosin activity could predict boar sperm freezability. For this purpose, we characterized the changes in sperm quality and acrosin activity throughout the cryopreservation procedure of sperm samples from 30 Pietrain boars by analyzing four critical steps: step 1 (extended sperm at 15 °C), step 2 (cooled sperm at 5 °C), step 3 (30 minutes postthaw), and step 4 (240 minutes postthaw). Freezability ejaculate groups were set on the basis of sperm motility and membrane integrity after freeze-thawing. Results obtained highlighted the low predictive value in terms of freezability of sperm motility and kinematics and sperm membrane integrity, as no differences between good and poor freezability ejaculates were seen before cryopreservation. Significant differences (P < 0.05) between ejaculate groups were observed in the cooling step at 5 °C for sperm kinetic parameters, and after thawing for sperm motility and membrane integrity. In contrast, acrosin activity appeared as an indicator of boar sperm freezability because the differences (P < 0.05) between good and poor freezability ejaculates manifested yet in extended samples at 15 °C. On the other hand, we also found that variations in sperm kinematics, membrane lipid disorder, intracellular calcium content, acrosome integrity, and acrosin activity throughout the cryopreservation procedure were indicative of a significant damage in spermatozoa during the cooling step in both ejaculate groups. In conclusion, the main finding of our study is that acrosin activity can be used as a reliable predictor of boar sperm freezability because it differs significantly between good and poor freezability ejaculates yet before freeze-thawing procedures took place, i.e., in the refrigeration step at 15 °C.

  18. Effects of bovine serum albumin on boar sperm quality during liquid storage at 17°C.

    PubMed

    Zhang, X-G; Yan, G-J; Hong, J-Y; Su, Z-Z; Yang, G-S; Li, Q-W; Hu, J-H

    2015-04-01

    This study aimed to investigate the effects of bovine serum albumin (BSA) on boar sperm quality during liquid storage at 17°C. Boar semen samples were collected and diluted with Modena containing different concentrations (0, 1, 2, 3, 4, 5 and 6 g/l) of BSA, and sperm motility, plasma membrane integrity, acrosome integrity, total antioxidative capacity (T-AOC) activity and malondialdehyde (MDA) content were measured and analysed. The results showed that Modena supplemented with 3, 4 and 5 g/l BSA could improve boar sperm motility, effective survival time and plasma membrane integrity (p < 0.05), decrease MDA content (p < 0.05), while no statistical difference was observed for sperm acrosome integrity and T-AOC activity among these three groups (p > 0.05). The semen sample diluted with Modena containing 4 g/l BSA could achieve optimum effect, and sperm survival time was 7.5 days. After 7 days preservation, sperm motility, plasma membrane integrity and acrosome integrity were 54%, 49% and 78%, respectively. T-AOC activity and MDA content were 1.03 U/ml and 17.5 nmol/ml, respectively. In conclusion, Modena supplemented with BSA reduced the oxidative stress and improved the sperm quality of boar semen during liquid storage at 17°C, and 4 g/l BSA was the optimum concentration. Further studies are required to obtain more concrete results on the determination of antioxidant capacities of BSA in liquid preserved boar semen.

  19. Membrane hyperpolarization during human sperm capacitation

    PubMed Central

    López-González, I.; Torres-Rodríguez, P.; Sánchez-Carranza, O.; Solís-López, A.; Santi, C.M.; Darszon, A.; Treviño, C.L.

    2014-01-01

    Sperm capacitation is a complex and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. Spermatozoa from several mammalian species, including mice, exhibit a capacitation-associated plasma membrane hyperpolarization, which is necessary for the acrosome reaction to occur. Despite its importance, this hyperpolarization event has not been adequately examined in human sperm. In this report we used flow cytometry to show that a subpopulation of human sperm indeed undergo a plasma membrane hyperpolarization upon in vitro capacitation. This hyperpolarization correlated with two other well-characterized capacitation parameters, namely an increase in intracellular pH and Ca2+ concentration, measured also by flow cytometry. We found that sperm membrane hyperpolarization was completely abolished in the presence of a high external K+ concentration (60 mM), indicating the participation of K+ channels. In order to identify, which of the potential K+ channels were involved in this hyperpolarization, we used different K+ channel inhibitors including charybdotoxin, slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K+ channel antagonists inhibited membrane hyperpolarization to a similar extent, suggesting that both members of the Slo family may potentially participate. Two very recent papers recorded K+ currents in human sperm electrophysiologically, with some contradictory results. In the present work, we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition, we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether, our data indicate that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm, where Slo3 channels are the main contributors to the

  20. Heparin-glutathione III: study with fluorescent probes as indicators of membrane status of bull sperm.

    PubMed

    Reyes, R; Martinez, J C; Delgado, N M; Merchant-Larios, H

    2002-01-01

    Sperm obtained from bull epididymes were used to validate in vitro the effect of heparin and reduced glutathione on sperm membrane status, with the use of sodium dodecyl sulfate (SDS) and Triton X-100 in the presence of propidium iodide (IP) and diacetate fluorescein (FDA). The metabolic activities of treated sperm were qualitatively monitored using an alamar Blue Redox fluorescence indicator. Heparin did not damage the sperm plasma membrane, whereas GSH and SDS at 26 h of incubation dissolved the plasma membrane and the acrosome. On the other hand, at time zero, Triton X-100 showed 75% of sperm stained with IP, indicating plasma membrane damage. Results validated by electron microscopy of thin sections of treated sperm showed complete lack of the membrane, acrosome, and postacrosomal membrane system with 0.01% Triton X-100. Extracellular 15 mM GSH completely disappeared the plasma membrane over the sperm nucleus, leaving the postacrosomal membrane system and nucleus without apparent damage. The metabolic activity was supported over 52 h of incubation in any of the incubation systems tested, including Triton X-100, which showed a spermaticide effect. The authors propose that membrane damage does not mean they are dead, no matter the vital stain employed, and also that FDA-IP staining can be used as a fluorescent marker of sperm plasmatic membrane permeabilization and nuclear swelling.

  1. Computational reacting gas dynamics

    NASA Technical Reports Server (NTRS)

    Lam, S. H.

    1993-01-01

    In the study of high speed flows at high altitudes, such as that encountered by re-entry spacecrafts, the interaction of chemical reactions and other non-equilibrium processes in the flow field with the gas dynamics is crucial. Generally speaking, problems of this level of complexity must resort to numerical methods for solutions, using sophisticated computational fluid dynamics (CFD) codes. The difficulties introduced by reacting gas dynamics can be classified into three distinct headings: (1) the usually inadequate knowledge of the reaction rate coefficients in the non-equilibrium reaction system; (2) the vastly larger number of unknowns involved in the computation and the expected stiffness of the equations; and (3) the interpretation of the detailed reacting CFD numerical results. The research performed accepts the premise that reacting flows of practical interest in the future will in general be too complex or 'untractable' for traditional analytical developments. The power of modern computers must be exploited. However, instead of focusing solely on the construction of numerical solutions of full-model equations, attention is also directed to the 'derivation' of the simplified model from the given full-model. In other words, the present research aims to utilize computations to do tasks which have traditionally been done by skilled theoreticians: to reduce an originally complex full-model system into an approximate but otherwise equivalent simplified model system. The tacit assumption is that once the appropriate simplified model is derived, the interpretation of the detailed numerical reacting CFD numerical results will become much easier. The approach of the research is called computational singular perturbation (CSP).

  2. PLCζ or PAWP: revisiting the putative mammalian sperm factor that triggers egg activation and embryogenesis.

    PubMed

    Kashir, Junaid; Nomikos, Michail; Swann, Karl; Lai, F Anthony

    2015-05-01

    In mammals, egg activation is initiated by multiple cytosolic Ca(2+) transients (Ca(2+) oscillations) that are triggered following delivery of a putative sperm factor from the fertilizing sperm. The identity of this 'sperm factor' thus holds much significance, not only as a vital component in creating a new life, but also for its potential therapeutic and diagnostic value in human infertility. Recent data have emerged suggesting the sperm factor may be a post-acrosomal sheath WW domain-binding protein (PAWP). However, a significant body of research points to a testis-specific phospholipase C zeta (PLCζ) as the sperm factor. Herein, we examine the evidence presented in favour of PAWP in relation to PLCζ and the requisite physiological properties of the mammalian sperm factor.

  3. The CatSper channel modulates boar sperm motility during capacitation.

    PubMed

    Vicente-Carrillo, Alejandro; Álvarez-Rodríguez, Manuel; Rodríguez-Martínez, Heriberto

    2017-03-01

    The cation channel of sperm (CatSper) comprises four transmembrane subunits specifically expressed in human, equine, murine and ovine spermatozoa, apparently implicated in capacitation, hyperactivation and acrosome exocytosis. Western blotting and immunocytochemistry showed hereby that CatSper subunits are also present in boar spermatozoa, primarily over the sperm neck, tail and cytoplasmic droplets; albeit CatSper -1 presented in addition some distribution over the membrane of the acrosome and CatSper -2 and -4 over the membrane of the post-acrosome. The role of the Catsper channel in boar spermatozoa was investigated by extending the spermatozoa in media containing different calcium (Ca(2+)) availability and exposure to the capacitation-trigger bicarbonate, to progesterone or CatSper inhibitors (Mibefradil and NNC 55-0396), separately or sequentially, at physiological and toxicological doses. Extracellular Ca(2+) availability, combined with bicarbonate exposure (capacitation-inducing conditions) decreased sperm motility, similarly to when spermatozoa incubated in capacitation-inducing conditions was exposed to Mibefradil and NNC 55-0396. Exposure of these spermatozoa to progesterone did not cause significant changes in sperm motility and nor did it revert its decrease induced by CatSper antagonists. In conclusion, the CatSper channel regulates sperm motility during porcine capacitation-related events in vitro. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

  4. Reduced sperm quality in relation to oxidative stress in red deer from a lead mining area.

    PubMed

    Reglero, Manuel M; Taggart, Mark A; Castellanos, Pilar; Mateo, Rafael

    2009-01-01

    We studied the effects of elevated heavy metal uptake on the sperm quality and the antioxidant mechanisms of sperm and testis of red deer from a Pb mining area in Spain. Testis, liver and bone of red deer from mining (n = 21) and control (n = 20) areas were obtained from hunters and analyzed for Pb, Zn, Cu, Cd, As and Se. Testes were weighed and measured. Motility, acrosome integrity and viability and functionality of membrane were evaluated in epididymal spermatozoa. Lipid peroxidation, total glutathione, glutathione peroxidase (GPX) and superoxide dismutase (SOD) were studied in testis and spermatozoa. Deer from mined areas showed less Cu in testis, a higher testis mass and size and reduced spermatozoa membrane viability and acrosome integrity. Effects on sperm quality were associated to decreased Cu and increased Se in testis, and to decreases in the activity of SOD and GPX in testis and spermatozoa.

  5. Influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection in pigs.

    PubMed

    García-Roselló, Empar; Matás, Carmen; Cánovas, Sebastián; Moreira, Pedro N; Gadea, Joaquín; Coy, Pilar

    2006-01-01

    The purpose of this study was to determine the influence of sperm pretreatment on the efficiency of intracytoplasmic sperm injection (ICSI) in pigs. This was done by examining the effect of 1) the conservation method (fresh vs frozen); 2) the sperm treatment preinjection (resuspension in Dulbecco phosphate-buffered saline (DPBS) vs selection by a Percoll gradient); and 3) the acrosomal and live or dead status of the spermatozoa (by incubation with or without calcium ionophore, 1 muM and 5 muM). In vitro matured porcine oocytes were injected with treated spermatozoa according to each experiment. All the experiments were done with non-artificially activated oocytes. The percentages of activation and cleavage were higher (68% vs 43% and 63% vs 43%, respectively, P < .05) in oocytes injected with fresh vs frozen spermatozoa. The DPBS treatment allowed higher cleavage proportions than the Percoll treatment (P < .05). Moreover, a boar effect was observed in the percentage of developing blastocysts. None of the studied parameters was affected by the acrosomal or the live or dead status of the spermatozoa injected. In conclusion, the use of fresh semen is recommended for porcine ICSI, as well as careful selection of the boar; Percoll treatment is only recommended for poor-quality samples or for removing toxic agents, and no exogenous form of activation or induction of the acrosome reaction is necessary for porcine oocytes to develop a male pronucleus and cleave up to the 2-cell stage after ICSI, although experimental conditions to reach the blastocyst stage need to be investigated further.

  6. Acrosome Reaction and Ca2+ Imaging in Single Human Spermatozoa: New Regulatory Roles of [Ca2+]i1

    PubMed Central

    Sánchez-Cárdenas, Claudia; Servín-Vences, Martha Rocio; José, Omar; Treviño, Claudia Lydia; Hernández-Cruz, Arturo; Darszon, Alberto

    2014-01-01

    ABSTRACT The spermatozoa acrosome reaction (AR) is essential for mammalian fertilization. Few methods allow visualization of AR in real time together with Ca2+ imaging. Here, we show that FM4-64, a fluorescent dye used to follow exocytosis, reliably reports AR progression induced by ionomycin and progesterone in human spermatozoa. FM4-64 clearly delimits the spermatozoa contour and reports morphological cell changes before, during, and after AR. This strategy unveiled the formation of moving tubular appendages, emerging from acrosome-reacted spermatozoa, which was confirmed by scanning electron microscopy. Alternate wavelength illumination allowed concomitant imaging of FM4-64 and Fluo-4, a Ca2+ indicator. These AR and intracellular Ca2+ ([Ca2+]i) recordings revealed that the presence of [Ca2+]i oscillations, both spontaneous and progesterone induced, prevents AR in human spermatozoa. Notably, the progesterone-induced AR is preceded by a second [Ca2+]i peak and ∼40% of reacting spermatozoa also manifest a slow [Ca2+]i rise ∼2 min before AR. Our findings uncover new AR features related to [Ca2+]i. PMID:25100708

  7. Conserved ram seminal plasma proteins bind to the sperm membrane and repair cryopreservation damage.

    PubMed

    Bernardini, A; Hozbor, F; Sanchez, E; Fornés, M W; Alberio, R H; Cesari, A

    2011-08-01

    Whole seminal plasma (SP) enhances the function and fertility of frozen/thawed ram sperm. The objective of the current study was to investigate whether SP proteins capable of binding to molecules from the sperm plasma membrane were conserved among ram breeds, and whether these proteins were sufficient to overcome cryopreservation-induced reductions in sperm quality. Whole ram SP, obtained from rams of various breeds, improved progressive motility of frozen/thawed sperm at all times evaluated (P < 0.05); however, it did not improve total motility (15 min, P = 0.480; 30 min, P = 0.764; and 45 min, P = 0.795). To identify SP proteins responsible for this effect, a new method was developed to retain SP proteins that bound specifically to the sperm membrane by immobilization of sperm membrane proteins. These proteins specifically bound to the sperm surface, especially the acrosomal region. Lactotransferrin, epididymal secretory protein E1, Synaptosomal-associated protein 29, and RSVP-20 were identified (mass spectrometry) in this fraction. The retained SP proteins fraction repaired ultrastructural damage of frozen/thawed sperm and, with the addition of fructose, significantly improved motility of frozen/thawed sperm. We concluded that SP proteins that bound to the sperm membrane were conserved among ram breeds, and that when added to frozen/thawed semen (along with an energy source), they repaired ram sperm damage and enhanced sperm motility. Copyright © 2011 Elsevier Inc. All rights reserved.

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

    PubMed

    Quevedo, María F; Lucchesi, Ornella; Bustos, Matías A; Pocognoni, Cristian A; De la Iglesia, Paola X; Tomes, Claudia N

    2016-10-28

    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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender.

    PubMed

    Pojprasath, T; Lohachit, C; Techakumphu, M; Stout, T; Tharasanit, T

    2011-06-01

    Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.

  10. Effects of different cryoprotective agents on ram sperm morphology and DNAintegrity.

    PubMed

    Nur, Z; Zik, B; Ustuner, B; Sagirkaya, H; Ozguden, C G

    2010-06-01

    This study investigates the effects of glycerol, 1,2 propanediol, sucrose, and trehalose on post-thaw motility, morphology, and genome integrity of Awassi ram semen. Ejaculates of thick consistency with rapid wave motion (>+++) and >70% initial motility were pooled. Sperm were diluted to a final concentration of 1/5 (semen/extender) in 0% cryoprotectant, 6% glycerol, 6% 1,2 propanediol, 62.5 mM sucrose or 62.5 mM trehalose using a two-step dilution method. The equilibrated semen was frozen in 0.25-ml straws. Semen samples were examined for sperm motility, defective acrosomes (FITC-Pisum sativum agglutinin (FITC PSA)), DNA integrity (acridine orange staining (AO)) and apoptotic activity (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and Caspase-3 activity) at four time points: after dilution with extender A, after cooling to 5 degrees C, after equilibration and post-thaw. Freezing and thawing procedures (cooling at 5 degrees C, dilution, equilibration, and thawing) had negative effects on motility (P<0.001), acrosome integrity (P<0.001), and DNA integrity as determined by AO (P<0.001) and TUNEL (P<0.001) assays. There were positive correlations between sperm with defective acrosomes and apoptotic (AO- and TUNEL-positive) spermatozoa. In contrast, a significant negative correlation was found between sperm motility and defective acrosomes and AO- and TUNEL positivity (P<0.01). The cryopreservation process acts as an apoptotic inducer in ram semen; all cryoprotectants used in the present study allowed apoptosis to some extent, with negative effects on sperm morphology and DNA integrity. The glycerol group performed better than the propanediol, sucrose, trehalose, and control groups in terms of post-thaw sperm motility but not DNA integrity. 2010 Elsevier Inc. All rights reserved.

  11. [Studies on the ploymorphic of sperm of F2 hybrids of red crucian carp x common carp].

    PubMed

    Li, Jian Zhong; Liu, Shao Jun; Zhang, Xuan Jie; Feng, Hao; Liu, Yun

    2004-08-01

    AThe ultrastructures of the sperm of F2 hybrids of red crucian carp x common carp were studied by using scanning and transmission electron microscope. The sperm of the F2 hybrids consisted of head, mid-piece and tail. There was no acrosome at the anterior end of the nuclears, whereas there was a vesicle. The results revealed that there existed obviously ploymorphic in the sperm of F2 hybrids. In the water-like semen from males of F2 hybrids, different sizes of the head of the sperm including haploid, diploid, tetraploid, and aneuploid sperm were observed. The head diameter of the smallest sperm was only 1.32 microm, but that of the biggest one was about 18.39 microm, and most of them varied from 1.85 to 2.15 microm. The haploid sperm was normal, while the a-neuploid, diploid, tetraploid and multiploid sperm were abnormal. Among the abnormal sperm, there was a super sperm with about 20 tails, whose head volume was much bigger than that of any other sperm. From the results of the transmission electron microscope, 3 sperm with two nucleus and 1 sperm with two tails were found. This study provided an useful evidence for the mechanism that the formation of tetraploid in F3 hybrids was due to the fertilization of the diploid eggs and diploid sperm produced by F2 hybrids.

  12. Reduction of the fertilizing capacity of sea urchin sperm by cannabinoids derived from marihuana. III. Activation of phospholipase A2 in sperm homogenate by delta 9-tetrahydrocannabinol.

    PubMed

    Chang, M C; Berkery, D; Laychock, S G; Schuel, H

    1991-07-25

    Inhibition of the egg jelly induced acrosome reaction by delta 9-tetrahydrocannabinol (THC) is associated with the localized disruption of the nuclear envelope and the formation of lipid deposits in sea urchin sperm. This suggests that THC may activate phospholipase(s) within the sperm. We now report effects of THC on phospholipase A2 activity in homogenates of sea urchin sperm using 1-stearoyl-2-[1-14C]arachidonyl phosphatidylcholine as substrate. The release of radioactive arachidonic acid was measured after a 30-min incubation with the enzyme. In the absence of exogenous Ca2+, 100 microM THC produced a significant (P less than 0.001) increase in phospholipase A2 activity. THC activated phospholipase A2 in a concentration (1-100 microM) and time-dependent (0-30 min) manner. Exogenous calcium (10 mM) significantly augmented basal (P less than 0.001) and THC-stimulated (P less than 0.005) phospholipase A2 activity. Calcium chelators [ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)] inhibited the basal level of phospholipase A2 activity in the sperm homogenate, and prevented the activation of phospholipase A2 by THC. Submicromolar levels of free calcium ions were required for THC stimulation of phospholipase A2. Cannabinol which mimics the effects of THC on the acrosome reaction also activated phospholipase A2 in sperm homogenate. These results suggest that THC may alter lipid metabolism in sperm by activating calcium-dependent phospholipase A2. Putative metabolites derived from this process may inhibit the acrosome reaction and thereby reduce the fertilizing capacity of sea urchin sperm.

  13. Influence of anaesthetic drugs on the epididymal sperm quality in domestic cats.

    PubMed

    Jiménez, E; Pérez-Marín, C C; Millán, Y; Agüera, E

    2011-02-01

    The present study investigated the effect of different anaesthetic agents commonly used in cats on the fresh and frozen-thawed epididymal sperm. Seventeen male domestic cats were castrated using pentobarbital, ketamine HCl or isoflurane. Sperm samples were recovered from epididymides and evaluated before and after freezing, determining the vigor, motility, morphology, acrosome status, sperm viability and functional membrane integrity. Fresh epididymal sperm was influenced by the drugs used, noting that motility features, i.e. vigor (p≤0.05) and progressive motility (p≤0.05), were higher for the inhalation anaesthetic while the others did not showed statistical differences. In frozen-thawed sperm samples, cats treated with barbiturics showed lower values for acrosome status (p≤0.05) and integrity and functionality of membrane (p≤0.05 and p≤0.01, respectively) than in the others groups. Results suggested that drugs used for castration in cats could affect the sperm quality and this should be considered when implementing sperm cryopreservation in the feline.

  14. Sperm-egg penetration assay assessment of the contraceptive effects of glycerol and egg yolk in rooster sperm diluents.

    PubMed

    Abouelezz, F M K; Castaño, C; Toledano-Díaz, A; Esteso, M C; López-Sebastián, A; Campo, J L; Santiago-Moreno, J

    2015-06-01

    Glycerol (GLY) and egg yolk (EY) are good cryoprotectants of avian and mammalian sperm, but in birds, they strongly inhibit the eventual fertilization of ova. Using the sperm penetration (SP-holes) assay and fertility trials, the present study investigates (1) the possible mechanism by which this contraceptive effect occurs in chickens and (2) the maximum concentrations of GLY and EY tolerated by fresh rooster sperm. Seventy Black-Barred Andaluza hens (five per treatment) were inseminated four times (twice per week) with 0.1 mL of fresh semen from roosters of the same breed diluted 1:1 (v:v) with Lake and Ravie medium containing different concentrations of GLY or EY. No adverse effects on acrosome integrity, sperm motility, or viability were seen with any concentration of GLY or EY. The number of SP-holes on perivitelline layer samples taken from above the germinal disc became progressively lower at GLY concentrations of 1.5% or greater (P > 0.05). No holes caused by sperms were seen in unfertilized eggs. The corresponding fertility results showed similar reductions when the GLY concentration was 1.5% or greater. No changes in the number of SP-holes were seen with increasing EY concentrations (0%-7.5%), nor were any differences in fertility observed, except for a reduction when 15% EY was used. The results therefore reveal that GLY affects the transit of sperms through the oviduct in their attempt to reach the infundibulum area, limiting their access to the ovum perivitelline layer. Egg yolk had no such effect, nor did it influence acrosome reaction capacity; its mechanism of contraceptive action therefore remains unknown. The maximum GLY and EY concentrations tolerated by the rooster sperm were 0.75% and 7.5%, respectively.

  15. Acrosome reaction is impaired in spermatozoa of obese men: a preliminary study.

    PubMed

    Samavat, Jinous; Natali, Ilaria; Degl'Innocenti, Selene; Filimberti, Erminio; Cantini, Giulia; Di Franco, Alessandra; Danza, Giovanna; Seghieri, Giuseppe; Lucchese, Marcello; Baldi, Elisabetta; Forti, Gianni; Luconi, Michaela

    2014-11-01

    To compare spontaneous (Sp-AR) and P-induced acrosome reaction (AR) in spermatozoa of obese and lean subjects. Bariatric unit at a university hospital. Prospective, observational study. Twenty-three obese (mean±SD body mass index [BMI], 44.3±5.9 kg/m2) and 25 age-matched lean (BMI, 24.2±3.0 kg/m2) subjects. None. Spontaneous and P-induced AR in spermatozoa of obese and lean subjects. A statistically significant difference was found between obese and lean cohorts in total T and calculated free T, E2, glycated hemoglobin, and high-density lipoproteins, whereas among the routine semen parameters analyzed, only immotile sperm percentage and ejaculate volume differed significantly. Spermatozoa of obese (n=13) vs. lean men (n=19) showed a higher Sp-AR (17.9%±7.2% vs. 8.3%±4.2%), which resulted in a reduced ability to respond to P evaluated as the AR-after-P-challenge parameter (3.5%±3.2% vs. 17.6%±9.2%). Multivariate analysis adjusted for age revealed a significant correlation between BMI, waist, E2, and glycated hemoglobin with both Sp-AR (age-adjusted r=0.654, r=0.711, r=0.369, and r=0.644, respectively) and AR-after-P-challenge (age-adjusted r=-0.570, r=-0.635, r=-0.507, and r=-0.563, respectively). A significant difference in sperm cholesterol content was reported between obese and lean men (29.8±19.5 vs. 19.1±14.6 ng/μg of proteins). Sperm AR is impaired in obese men, showing reduced response to P and elevated Sp-AR, associated with altered circulating levels of E2 and sperm cholesterol content. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. Tales of the Tail and Sperm Head Aches Changing concepts on the prognostic significance of sperm pathologies affecting the head, neck and tail

    PubMed Central

    Chemes, Héctor E; Alvarez Sedo, Cristian

    2012-01-01

    This article presents an update on the variable prognostic significance of different sperm pathologies in patients with severe male factor infertility due to morphology and motility disorders. Severe asthenozoospermia is one of the leading causes of male infertility as spermatozoa cannot reach the oocyte and/or penetrate normally. Identifying structural causes of sperm immotility was of great concern before the advent of intracytoplasmic sperm injection (ICSI), because immotility was the limiting factor in the treatment of these patients. In these cases, in vitro methods are used to identify live spermatozoa or stimulate sperm motility to avoid selection of non-viable cells. With these advances, fertilization and pregnancy results have improved dramatically. The identification of genetic phenotypes in asthenozoospermia is important to adequately inform patients of treatment outcomes and risks. The one sperm characteristic that seriously affects fertility prognosis is teratozoospermia, primarily sperm head and neck anomalies. Defects of chromatin condensation and acrosomal hypoplasia are the two most common abnormalities in severe teratozoospermia. The introduction of microscopic methods to select spermatozoa and the development of new ones to evaluate sperm quality before ICSI will assure that ultrastructural identification of sperm pathologies will not only be of academic interest, but will also be an essential tool to inform treatment choice. Herein, we review the differential roles played by sperm components in normal fertilization and early embryo development and explore how assisted reproductive technologies have modified our concepts on the prognostic significance of sperm pathologies affecting the head, neck, mid-piece and tail. PMID:22198630

  17. Sperm morphology of Muscidifurax uniraptor (Hymenoptera: Chalcidoidea: Pteromalidae).

    PubMed

    Santos, Helen Pinto; Barcellos, Marcelo Silva; Reis, Aline Beatriz; Dolder, Heidi; Lino-Neto, José

    2016-05-01

    Sperm morphology of the parasitoid Muscidifurax uniraptor was investigated under light and transmission electron microscopy. M. uniraptor sperm are filiform, spiraled, approximately 150 μm in length, with a distinctive head, hooded by an extracellular sheath and a flagellum. This extracellular layer, from which many filaments radiate, measures approximately 90 nm in thickness and covers a small acrosome and the anterior nuclear region. The acrosome is composed of an acrosomal vesicle and a perforatorium with its base inserted in the nuclear tip. The nucleus is filled with homogeneously compacted chromatin. The centriolar adjunct extends towards the anterior portion in a spiral around the nucleus for 3.5 μm in length. The two mitochondrial derivatives begin exactly at the centriole adjunct base and, in cross-section, have a circular shape with equal areas that are smaller than the axoneme diameter. It is coiled, with 9 + 9 + 2 microtubules and begins from the centriole, just below the nuclear base. The axoneme is connected to the mitochondrial derivatives by two small irregularly shaped masses. Between the derivatives and the axoneme, the 'center-flagellar material' is observed. Overall, these characteristics are recognized in other Chalcidoidea, especially in the eurytomids, but together they form a set of species-specific data.

  18. The secretory products of Trichomonas vaginalis decrease fertilizing capacity of mice sperm in vitro.

    PubMed

    Roh, Jaesook; Lim, Young-Su; Seo, Min-Young; Choi, Yuri; Ryu, Jae-Sook

    2015-01-01

    Trichomonas vaginalis infection is one of the most prevalent sexually transmitted infections in humans and is now recognized as an important cause of infertility in men. There is little information about the effect of extracellular polymeric substances (EPS) from T. vaginalis on sperm, but previous reports do not provide a conclusive description of the functional integrity of the sperm. To investigate the impact of EPS on the fertilizing capacity of sperm, we assessed sperm motility, acrosomal status, hypo-osmotic swelling, and in vitrofertilization rate after incubating the sperm with EPS in vitrousing mice. The incubation of sperm with EPS significantly decreased sperm motility, viability, and functional integrity in a concentration and time-dependent manner. These effects on sperm quality also resulted in a decreased fertilization rate in vitro. This is the first report that demonstrates the direct negative impact of the EPS of T. vaginalis on the fertilization rate of sperm in vitro. However, further study should be performed using human sperm to determine if EPS has similar negative impact on human sperm fertilizing capacity in vitro.

  19. The secretory products of Trichomonas vaginalis decrease fertilizing capacity of mice sperm in vitro

    PubMed Central

    Roh, Jaesook; Lim, Young-Su; Seo, Min-Young; Choi, Yuri; Ryu, Jae-Sook

    2015-01-01

    Trichomonas vaginalis infection is one of the most prevalent sexually transmitted infections in humans and is now recognized as an important cause of infertility in men. There is little information about the effect of extracellular polymeric substances (EPS) from T. vaginalis on sperm, but previous reports do not provide a conclusive description of the functional integrity of the sperm. To investigate the impact of EPS on the fertilizing capacity of sperm, we assessed sperm motility, acrosomal status, hypo-osmotic swelling, and in vitro fertilization rate after incubating the sperm with EPS in vitro using mice. The incubation of sperm with EPS significantly decreased sperm motility, viability, and functional integrity in a concentration and time-dependent manner. These effects on sperm quality also resulted in a decreased fertilization rate in vitro. This is the first report that demonstrates the direct negative impact of the EPS of T. vaginalis on the fertilization rate of sperm in vitro. However, further study should be performed using human sperm to determine if EPS has similar negative impact on human sperm fertilizing capacity in vitro. PMID:25578937

  20. Validation of a laboratory‐developed test of human sperm capacitation

    PubMed Central

    Moody, Melissa A.; Cardona, Cristina; Simpson, Alana J.; Smith, T. Timothy; Travis, Alexander J.

    2017-01-01

    Sperm must undergo capacitation to become fertilization competent. Here we validated that monosialotetrahexosylganglioside (GM1) localization patterns, which were assessed in the Cap‐Score™ Sperm Function Test, reflect a capacitated state in human sperm. First, we defined patterns representing sperm that do or do not respond to stimuli for capacitation. Sperm with “capacitated” patterns had exposed acrosomal carbohydrates and underwent acrosome exocytosis in response to calcium ionophore (A23187). Precision was evaluated by percent change of the Cap‐Score measured for 50, 100, 150, and 200 sperm. Changes of 11%, 6%, and 5% were observed (n ≥ 23); therefore, we counted ≥150 sperm per condition. Variance within and between readers was evaluated using 20 stitched image files generated from unique ejaculates. Two trained readers randomly resampled each image 20 times, reporting an average standard deviation of 3 Cap‐Score units and coefficient of variation of 13% when rescoring samples, with no difference between readers. Semen liquefaction times ≤2 hr and mechanical liquefaction with Pasteur or wide‐orifice transfer pipettes did not alter Cap‐Score values. However, liquefaction with chymotrypsin (p = 0.002) and bromelain (p = 0.049) reduced response to capacitating stimuli and induced membrane damage, while counterintuitively improving sperm motility. Together, these data validate the Cap‐Score assay for the intended purpose of providing information on sperm capacitation and male fertility. In addition to its clinical utility as a diagnostic tool, this test of sperm function can reveal the impact of common practices of semen handling on the ability of sperm to respond to capacitation stimuli. PMID:28418600

  1. Boar seminal plasma exosomes: effect on sperm function and protein identification by sequencing.

    PubMed

    Piehl, Lidia L; Fischman, M Laura; Hellman, Ulf; Cisale, Humberto; Miranda, Patricia V

    2013-04-15

    Mammalian seminal plasma contains membranous vesicles (exosomes), with a high content of cholesterol and sphingomyelin and a complex protein composition. Their physiological role is uncertain because sperm stabilization and activation effects have been reported. To analyze a putative modulatory role for semen exosomes on sperm activity in the boar, the effects of these vesicles on several sperm functional parameters were examined. Additionally, boar exosome proteins were sequenced and their incorporation into sperm was explored. Boar sperm were incubated under conditions that induce capacitation, manifested as increased tyrosine phosphorylation, cholesterol loss and greater fluidity in apical membranes, and the ability to undergo the lysophosphatidylcholine-induced acrosome reaction. After establishing this cluster of capacitation-dependent functional parameters, the effect produced by exosomes when present during or after sperm capacitation was analyzed. Exosomes inhibited the capacitation-dependent cholesterol efflux and fluidity increase in apical membranes, and the disappearance of a 14-kD phosphorylated polypeptide. In contrast, the acrosome reaction (spontaneous and lysophosphatidylcholine-induced) was not affected, and sperm binding to the oocyte zona pellucida was reduced only when vesicles were present during gamete coincubation. Liposomes with a lipid composition similar to that present in exosomes mimicked these effects, except the one on zona pellucida binding. Interaction between exosomes and sperm was confirmed by transfer of aminopeptidase activity. In addition, the major exosome protein, identified as actin, appeared to associate with sperm after coincubation. Exosome composition had a predominance for structural proteins (actin, plastin, ezrin, and condensin), enzymes, and several porcine seminal plasma-specific polypeptides (e.g., spermadhesins). Transfer of proteins from exosome to sperm and their ability to block cholesterol efflux supports a

  2. Sperm-surface ATP in boar spermatozoa is required for fertilization: relevance to sperm proteasomal function.

    PubMed

    Yi, Young-Joo; Park, Chang-Sik; Kim, Eui-Sook; Song, Eun-Sook; Jeong, Ji-Hyeon; Sutovsky, Peter

    2009-01-01

    Extracellular ATP has been implicated in a number of cellular events, including mammalian sperm function. The complement of ATP-dependent sperm proteins includes six subunits of the 26S proteasome, a multi-subunit protease specific to ubiquitinated substrate-proteins. Proteolysis of ubiquitinated proteins by the 26S proteasome is necessary for the success of mammalian fertilization, including but not limited to acrosomal exocytosis (AE) and sperm-zona pellucida (ZP) penetration. The 26S proteasome is uniquely present on the sperm acrosomal surface during mammalian, ascidian, and invertebrate fertilization. The proteasome is a multi-subunit protease complex of approximately 2 MDa composed of the 19S regulatory complex and a 20S proteolytic core. Integrity of the 19S complex is maintained by six 19S ATPase subunits (PSMC1 through PSMC6). Consequently, we hypothesized that fertilization will be blocked by the depletion of sperm-surface associated ATP (ssATP). Depletion of ssATP by the Solanum tuberosum apyrase, a 49 kDa, non-cell permeant enzyme, significantly reduced the ATP content measured by an adapted luminescence-ATP assay from which all permeabilizing agents were excluded. Addition of active apyrase to porcine in vitro fertilization (IVF) medium caused a concentration dependent reduction in the overall fertilization rate. No such outcomes were observed in control groups using heat-inactivated apyrase. Apyrase treatment altered the band pattern of 19S ATPase subunits PSMC1 (Rpt2) and PSMC4 (Rpt3) in Western blotting, suggesting that it had an effect on the integrity of the sperm proteasomal 19S complex. Apyrase only altered the proteasomal core activities slightly, since these activities are not directly dependent on external ATP. In contrast, sperm treatment with MG132, a specific inhibitor of the proteasomal core chymotrypsin-like activity, inhibited the target proteolytic activity, but also induced a compensatory elevation in proteasomal peptidyl

  3. Porcine sperm vitrification I: cryoloops method.

    PubMed

    Arraztoa, C C; Miragaya, M H; Chaves, M G; Trasorras, V L; Gambarotta, M C; Péndola, C H; Neild, D M

    2016-09-29

    The aims of this study were to evaluate porcine sperm vitrification in cryoloops, with and without two different cryoprotectants and assess two warming procedures. Extended (n = 3; r = 4) and raw (n = 5; r = 2) semen was diluted in media without and with cryoprotectants (4% dimethylformamide and 4% glycerol) to a final concentration of 20 × 10(6) spermatozoa ml(-1) and vitrified using the cryoloops method. Two warming procedures were evaluated: rapid method (30 s at 37°C) and an ultra-rapid method (7 s at 75°C, followed by 30 s at 37°C). Total motility (phase contrast), sperm viability (6-carboxifluorescein diacetate and propidium iodide stain), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated before and after vitrification and analysed using Friedman's test. In all media, the only seminal parameters that were maintained after vitrification were chromatin condensation and integrity. Vitrification of porcine spermatozoon using cryoloops, both in the presence or absence of cryoprotectants and independent of the warming procedure used, permits conservation of sperm chromatin condensation and integrity. It would be interesting to further verify this by producing porcine embryos using vitrified spermatozoon with intracytoplasmic sperm injection.

  4. Ultrastructural Morphology of Sperm from Human Globozoospermia.

    PubMed

    Ricci, Giuseppe; Andolfi, Laura; Zabucchi, Giuliano; Luppi, Stefania; Boscolo, Rita; Martinelli, Monica; Zweyer, Marina; Trevisan, Elisa

    2015-01-01

    Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia.

  5. Ultrastructural Morphology of Sperm from Human Globozoospermia

    PubMed Central

    Ricci, Giuseppe; Andolfi, Laura; Zabucchi, Giuliano; Luppi, Stefania; Boscolo, Rita; Martinelli, Monica; Zweyer, Marina; Trevisan, Elisa

    2015-01-01

    Globozoospermia is a rare disorder characterized by the presence of sperm with round head, lacking acrosome. Coiling tail around the nucleus has been reported since early human studies, but no specific significance has conferred it. By contrast, studies on animal models suggest that coiling tail around the nucleus could represent a crucial step of defective spermatogenesis, resulting in round-headed sperm. No observations, so far, support the transfer of this hypothesis to human globozoospermia. The purpose of this work was to compare ultrastructural morphology of human and mouse model globozoospermic sperm. Sperm have been investigated by using scanning and transmission electron microscopy. The images that we obtained show significant similarities to those described in GOPC knockout mice, an animal model of globozoospermia. By using this model as reference, we were able to identify the probable steps of the tail coiling process in human globozoospermia. Although we have no evidence that there is the same pathophysiology in man and knocked-out mouse, the similarities between these ultrastructural observations in human and those in the experimental model are very suggestive. This is the first demonstration of the existence of relevant morphological homologies between the tail coiling in animal model and human globozoospermia. PMID:26436098

  6. Pre-treatment of sperm reduces success of ICSI in the pig.

    PubMed

    Nakai, Michiko; Ito, Junya; Sato, Ken-Ichi; Noguchi, Junko; Kaneko, Hiroyuki; Kashiwazaki, Naomi; Kikuchi, Kazuhiro

    2011-08-01

    In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.

  7. Unexpected Flagellar Movement Patterns and Epithelial Binding Behavior of Mouse Sperm in the Oviduct1

    PubMed Central

    Chang, Haixin; Suarez, Susan S.

    2012-01-01

    ABSTRACT In order to better understand how sperm movement is regulated in the oviduct, we mated wild-type female mice with Acr-EGFP males that produce sperm with fluorescent acrosomes. The fluorescence improved our ability to detect sperm within the oviduct. Oviducts were removed shortly before or after ovulation and placed in chambers on a warm microscope stage for video recording. Hyperactivated sperm in the isthmic reservoir detached frequently from the epithelium and then reattached. Unexpectedly, most sperm found in the ampulla remained bound to epithelium throughout the observation period of several minutes. In both regions, most sperm produced deep flagellar bends in the direction opposite the hook of the sperm head. This was unexpected, because mouse sperm incubated under capacitating conditions in vitro primarily hyperactivate by producing deep flagellar bends in the same direction as the hook of the head. In vitro, sperm that are treated with thimerosal to release Ca2+ from internal stores produce deep anti-hook bends; however, physical factors such as viscous oviduct fluid could also have influenced bending in oviductal sperm. Some sperm detached from epithelium in both the ampulla and isthmus during strong contractions of the oviduct. Blockage of oviduct contractions with nicardipine, however, did not stop sperm from forming a storage reservoir in the isthmus or prevent sperm from reaching the ampulla. These observations indicate that sperm continue to bind to oviductal epithelium after they leave the isthmic reservoir and that sperm motility is crucial in the transport of sperm to the fertilization site. PMID:22337334

  8. The role of sperm proteasomes during sperm aster formation and early zygote development: implications for fertilization failure in humans.

    PubMed

    Rawe, Vanesa Y; Díaz, Emilce S; Abdelmassih, Roger; Wójcik, Cezary; Morales, Patricio; Sutovsky, Peter; Chemes, Héctor E

    2008-03-01

    BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.

  9. Factor H in Porcine Seminal Plasma Protects Sperm against Complement Attack in Genital Tracts*

    PubMed Central

    Sakaue, Tomohisa; Takeuchi, Keisuke; Maeda, Toshinaga; Yamamoto, Yoshio; Nishi, Katsuji; Ohkubo, Iwao

    2010-01-01

    We found that factor H (FH) exists in porcine seminal plasma. Purified FH strongly inhibited serum alternative pathway complement activation against lipopolysaccharide. The molecular weight, pI, and heparin-binding activity of the purified protein were different from those of purified FH from porcine serum. The complement regulatory activity of seminal plasma FH was ∼2-fold stronger than that of serum FH. Treatment of purified serum FH with sialidase and N-glycosidase F gave almost the same results as those of seminal plasma FH. The deletion of sialic acid from the carbohydrate chains of both FHs contributed to heparin-binding and complement regulatory activities. Results of reverse transcriptase-PCR, Western blot analysis, and immunohistochemistry showed that seminal plasma FH is mainly secreted from epithelial cells of the seminal vesicle in male genital tracts. FH was also detected in the outer acrosomal region of ejaculated sperm by immunofluorescence staining, and found that the purified FH from the sperm membrane has the same complement regulatory activity as that of seminal plasma FH. The ejaculated sperm possessing FH in the outer acrosomal region considerably evaded complement attack. We also found that there is strong complement activity in fluids from female genital tract ducts. These findings indicate that FH bound to the outer acrosomal region and soluble FH play important roles in protecting sperm against complement attack in male and female genital tracts. PMID:19920146

  10. An in vitro study of the effect of mifepristone and ulipristal acetate on human sperm functions.

    PubMed

    Ko, J K Y; Huang, V W; Li, R H W; Yeung, W S B; Ho, P C; Chiu, P C N

    2014-11-01

    Ulipristal acetate (UPA) and mifepristone are currently well-established agents for emergency contraception. Both drugs are selective progestogen receptor modulators which have been shown to have better efficacy than the widely used levonorgestrel in prevention of pregnancy. However, there is only limited information on the action of UPA on sperm function. The present study compared the in vitro biological effects of mifepristone and UPA on human sperm functions. Spermatozoa from semen samples with normal semen parameters were isolated. Capacitated spermatozoa were pre-incubated with 0.04, 0.4, 4 and 40 μM mifepristone or UPA for 1 h. Sperm motility, viability, DNA integrity, capacitation, spontaneous acrosome reaction, spontaneous hyperactivation, zona pellucida (ZP) binding capability and intracellular calcium concentration ([Ca(2+)]i) were determined. The effects of mifepristone and UPA on progesterone-induced acrosome reaction, hyperactivation and [Ca(2+)]i were also studied. Our results showed that mifepristone and UPA dose-dependently suppressed progesterone-induced acrosome reaction, hyperactivation and [Ca(2+)]i at concentrations ≥0.4 μM in human spermatozoa. Both compounds did not affect sperm motility, viability, DNA integrity, capacitation, spontaneous acrosome reaction, spontaneous hyperactivation, ZP binding capability and [Ca(2+)]i. This study demonstrated that UPA and mifepristone modulate human sperm functions by acting as progesterone antagonists. The results enable us to gain a better understanding of the mechanisms by which mifepristone and UPA work for emergency contraception, and provide a scientific basis for their clinical application. © 2014 American Society of Andrology and European Academy of Andrology.

  11. The mu (μ) and delta (δ) opioid receptors modulate boar sperm motility.

    PubMed

    Vicente-Carrillo, Alejandro; Álvarez-Rodríguez, Manuel; Rodríguez-Martínez, Heriberto

    2016-08-01

    Endogenous and exogenous opioids modulate reproductive functions in target cells via opioid receptors (μ, δ, and κ). Sperm motility is a metric of gamete functionality, and serves as a suitable parameter for in vitro drug-induced toxicity assays. This study identifies the presence and location of opioid receptors in pig spermatozoa as well as their functional response after in vitro challenge with known agonists (morphine [μ]; [D-Pen 2,5]-enkephanile [δ]; and U 50488 [κ]) and antagonists (naloxone [μ]; naltrindole [δ]; and nor-binaltrorphimine [κ]). Only the μ- and δ-opioid receptors were present in the boar sperm plasma membrane, overlying the acrosome, neck, and principal piece. Challenge experiments with agonists and antagonists identified both μ- and δ-opioid receptors as regulators of sperm kinematics, wherein μ maintains or increases sperm movement whereas δ decreases sperm motility over time. Mol. Reprod. Dev. 83: 724-734, 2016 © 2016 Wiley Periodicals, Inc.

  12. The SLO3 sperm-specific potassium channel plays a vital role in male fertility

    PubMed Central

    Santi, Celia M; Martínez-López, Pablo; de la Vega-Beltrán, José Luis; Butler, Alice; Alisio, Arturo; Darszon, Alberto; Salkoff, Lawrence

    2010-01-01

    Here we show a unique example of male infertility conferred by a gene knock-out of the sperm-specific, pH-dependent SLO3 potassium channel. In striking contrast to wild-type sperm which undergo membrane hyperpolarization during capacitation, we found that SLO3 mutant sperm undergo membrane depolarization. Several defects in SLO3 mutant sperm are evident under capacitating conditions, including impaired motility, a bent “hairpin” shape, and failure to undergo the acrosome reaction (AR). The failure of AR is rescued by valinomycin which hyperpolarizes mutant sperm. Thus SLO3 is the principal potassium channel responsible for capacitation-induced hyperpolarization, and membrane hyperpolarization is crucial to the AR. PMID:20138882

  13. Prognosis for sperm fertilizability: analysis of different variables in men.

    PubMed

    Check, J H; Check, M L; Katsoff, D

    2002-01-01

    An overview of various sperm tests is presented. The standard semen analysis obtained by most clinicians evaluating infertility usually consists of sperm concentration, percent motility, quality of motility, and sperm morphology. Unfortunately, unless the motile density is extremely low, the count and motility are not good prognosticators of fertility potential. Values above the norm for normal fertile couples unfortunately cannot reliably predict normal fertility potential. It is important to find sperm tests that are easy to perform, are relatively inexpensive, and provide an accurate prognosis. Strict morphology was hoped to be such a tool with initial optimism that it was far superior to standard morphology. Unfortunately, this test also failed to be the ideal inexpensive prognostic test after further evaluation. One test that is inexpensive and highly correlates with fertilizability is the presence of antisperm antibodies since their presence frequently does not alter count, motility, or morphology. This test should be performed as part of the routine semen analysis. Other tests highly correlate with the achievement of pregnancy and are simple and inexpensive to perform, but, interestingly, do not correlate with fertilizability. These include the hypoosmotic swelling test (HOST) and the sperm stress test. Abnormalities in these tests imply a different abnormality of sperm that leads to conception failure and that is the transfer of a toxic factor from the sperm to oocyte to embryo that prevents the embryo from implanting. Certainly, the simple, inexpensive HOST should be performed routinely. Other tests of sperm function, e.g., sperm penetration assay, sperm zona pellucida binding assay, and acrosome reaction, have their definite place in the evaluation of the infertile male. However, because they are expensive and difficulty to perform they lend themselves to certain specific circumstances but not to routine testing.

  14. The osmotic tolerance of boar spermatozoa and its usefulness as sperm quality parameter.

    PubMed

    Yeste, Marc; Briz, Mailo; Pinart, Elisabeth; Sancho, Sílvia; Bussalleu, Eva; Bonet, Sergi

    2010-06-01

    Predicting the fertility outcome of ejaculates is very important in the field of porcine reproduction. The aims of this study were to determine the effects of different osmotic treatments on boar spermatozoa and to correlate them with fertility and prolificacy, assessed as non-return rates within 60 days (NRR(60d)) of the first inseminations, and litter size (LS), respectively. Sperm samples (n=100) from one hundred healthy Piétrain boars were used to assess 48 treatments combining different osmolalities (ranged between 100 and 4000 mOsm kg(-1)), different compounds used to prepare anisotonic solutions, and two different modalities: return and non-return to isotonic conditions. Sperm quality was evaluated before and after applying the treatments on the basis of analyses of sperm viability, motility, morphology and percentages of acrosome-intact spermatozoa. Statistical analyses were performed using a one-way ANOVA and post hoc Tukey's test, linear regression analyses (Pearson correlation and multiple regression) and Jackknife cross-validation. Although three conventional parameters: sperm viability, sperm morphology and the percentages of acrosome-intact spermatozoa were significantly correlated with NRR(60d) and with LS, their respective osmotic tolerance parameters (defined for each parameter and treatment regarding with negative control) presented a higher Pearson coefficient with both fertility and prolificacy in three treatments (150 mOsm kg(-1) with non-return to isotonic conditions, 200 mOsm kg(-1) with return and 500 mOsm kg(-1) using sodium citrate and non-return to isotonic conditions). We conclude that osmotic resistance in sperm viability, sperm morphology and acrosome-intactness in the treatments mentioned above could be assessed along with classical parameters to better predict the fertilising ability of a given ejaculate.

  15. Tmvoc-React

    SciTech Connect

    Xu, Tianfu

    2008-02-12

    The TMVOC-REACT simulator was generated by replacing the fluid and heat flow part, TOUGH2, in TOUGHREACT with TMVOC. Both programs have been distributed to the public through the US Department of Energy's Energy Science and Technology Software Center. TMVOC is a program for three-phase non-isothermal flows of multi-component hydrocarbon mixtures in variably saturated heterogeneous media. TMVOC was initially designed for studying subsurface contamination by volatile organic compounds (VOCs), such as hydrocarbon fuels and industrial solvents. It can model the one-, two-, or three-dimensional migration of non-aqueous phase liquids (NAPLs) through the unsaturated and saturated zones, the formation of an oil lens on the water table, the dissolution and subsequent transport of VOCs in groundwater, as well as the vaporization and migration of VOCs in the interstitial air of the unsaturated zone. TOUGHREACT is a numerical simulation program for chemically reactive nonisothermal flows of multiphase fluids in porous and fractured media. A variety of subsurface thermo-physical-chemical processes can be considered under a wide range of conditions of pressure, temperature, water saturation, ionic strength, pH and Eh. Intractions between mineral assemblages and fluids can occur under local equilibrium or kinetic rates. The gas phase can be chemically active. Precipitation and dissolution reactions can change formation porosity and permeability. The program can be applied to many geologic systems and environmental problems, including geothermal systems, diagenetic and weathering processes, subsurface waste disposal, aid mine drainage remediation, and contaminant transport.

  16. Cryopreservation of Cynomolgus Macaque (Macaca fascicularis) Sperm by Using a Commercial Egg-YolkFree Freezing Medium.

    PubMed

    Yan, Yaping; Ao, Lei; Wang, Hong; Duan, Yanchao; Chang, Shaohui; Chen, Bingbing; Zhi, Dalong; Li, Sujuan; Niu, Yuyu; Ji, Weizhi; Si, Wei

    2016-11-01

    Conventional TRISegg yolk (TEY) freezing medium for the cryopreservation of NHP sperm has the risk of contamination due to widespread zoonotic diseases. This study was aimed at determining the optimal glycerol concentration, freezing rate, and holding time in liquid N2 vapor for the cryopreservation of cynomolgus macaque sperm by using a commercial egg-yolkfree freezing medium (SC medium) designed for human sperm cryopreservation. Sperm motility and acrosomal integrity after freezing were assessed. Sperm in SC medium (dilution ratio, 3:1) frozen at cooling rates of 67 and 183C/min in liquid N2 vapor showed higher post-thaw motility than did samples frozen at 435C/min. At the cooling rate of 183C/min and dilution in SC medium at a 3:1 ratio, post-thaw motility was higher after a holding time of 10 min than after 30 min (recommended by the manufacturer). In addition, post-thaw motility of sperm frozen in SC medium was higher with dilution ratios of 3:1, 4.5:1, and 6:1 compared with 9:1, 10.5:1, and 12:1, and the sample diluted 12:1 showed the lowest percentage of thawed sperm with intact acrosomes. Sperm showed higher post-thaw motility after freezing in TEY than in SC medium; acrosomal integrity did not differ between the 2 media. Our results indicated that cynomolgus macaque sperm can be cryopreserved successfully by using a commercial egg-yolkfree freezing medium, which provides an option for genetic preservation with decreased zoonotic risk in this important NHP species.

  17. Integrity of head and tail plasmalemma is associated with different kinetic variables in boar sperm.

    PubMed

    Grieblová, Adéla; Pintus, Eliana; Ros-Santaella, José Luis

    2017-09-01

    An intact and functional sperm plasmalemma has a major role in sperm motility and fertilizing capacity. Several techniques have been developed to evaluate the integrity of the sperm plasma membrane, but there are still some inconsistencies concerning the methods that are more closely associated with sperm function. In this study, the aim was to: i) evaluate the integrity of the boar sperm plasmalemma during 72h of semen storage at 17°C using four techniques: eosin/nigrosin (E/N), propidium iodide/carboxyfluorescein diacetate (PI/CFDA), hypo-osmotic swelling test (HOST), and combined HOST with eosin staining (HOST/E), ii) assess the correlations and the limits of consistency among these techniques, iii) and estimate the relationships with the acrosomal status and sperm kinetics. Results indicate that the integrity of the sperm plasmalemma decreases during 72h of storage, although significant differences were found only using the HOST and HOST/E techniques. Moreover, use of E/N and PI/CFDA results in greater values relative to the undamaged sperm membrane than use of HOST and HOST/E at any incubation time. Overall, results using all techniques were consistent and correlate except for findings with PI/CFDA and HOST, which was slightly below 95%. Moreover, values using the techniques for the evaluation of the integrity of the sperm head and tail membranes are positively associated with the acrosomal status and different kinetic variables with the tail integrity being related to rapid linear trajectories and the head integrity to rapid curvilinear trajectories. The results of this study provide new insights into the relevance of evaluating the boar sperm plasmalemma in the routine spermiogram. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Spermatogenesis and sperm ultrastructure in the polychaete genus Ophryotrocha (Dorvilleidae)

    NASA Astrophysics Data System (ADS)

    Pfannenstiel, Hans-Dieter; Grünig, Charlotte

    1990-06-01

    The details of spermatogenesis and spermiogenesis are described for Ophryotrocha puerilis. The ultrastructure of mature sperm is shown for O. puerilis, O. hartmanni, O. gracilis, O. diadema, O. labronica, and O. notoglandulata. Clusters of sixteen cells each are proliferated by two stem cells in each setigerous segment of O. puerilis representing the very early stages of both oogenesis and spermatogenesis. In each spermatocyte-I cluster, the cells are interconnected by cytoplasmic bridges. Early, clusters are enveloped by peritoneal sheath cells. These transient gonad walls break down prior to meiosis. The meiotic processes may start in the clusters with the cells still interconnected, or during breakdown of the original cluster, giving rise to smaller subclusters of both spermatocytes I and spermatocytes II with various numbers of cells. Finally, spermatid tetrads are present. As spermiogenesis progresses, the tetrads disintegrate. Golgi vesicles in both spermatocytes and spermatids contain electron-dense material, presumably preacrosomal. The acrosome is formed by such vesicles. In the six species studied here, the acrosomes appear to be of a similar overall structure but are of different shape. Centrioles are usually located beneath the acrosome. The distal centriole forms the basal body of a flagellum-like cytoplasmic process. The microtubules of these flagellar equivalents do not show a normal ciliar arrangement. The flagellar equivalent appears to be non-motile. In O. hartmanni and in O. notoglandulata, a flagellar equivalent is missing. Microtubules originating from the proximal end of the distal centriole stretch to the nuclear envelope. This feature appears to be especially conspicuous in O. puerilis and in O. labronica. In O. labronica and in O. notoglandulata, bundles of microtubules paralleling the cell perimeter appear to stabilise the sperm. Various numbers of mitochondria are either randomly distributed around the nucleus or accumulate on one side

  19. Acrosomal Component of Rat Round Spermatids Recognized by a Novel Monoclonal Antibody.

    PubMed

    Russinova; Atanassova; Paskaleva; Kancheva

    1998-09-01

    OBJECTIVE: To characterize immunocytochemically the antigen recognized which appears at specific stages of germ cell development and acrosomal biogenesis by the novel monoclonal antibody (Mab 3C2). METHODS: The novel monoclonal antibody (Mab 3C2) raised against testicular Sertoli and germ cells. RESULTS: The immunoreactivity of this Mab in testicular sections from immature 20-day-old rats was confined to the pachytene spermatocytes. In adult testis the Mab 3C2, besides meiotic cells, recognized also acrosomal component of round spermatids. The immune reaction was observed in Golgi and cap phases of acrosomal development until the stage VIII of the cycle of the seminiferous epithelium. Immunostaining was absent in acrosome of elongating and mature spermatids and indicated that some modifications in acrosomal protein may exist in subsequent stages of acrosomal development. CONCLUSIONS: Novel Mab 3C2 shares a common antigen in pachytene spermatocytes and round spermatids. Therefore, it may be a marker of meiotic and postmeiotic germ cells.

  20. Role and Regulation of Sperm Gelsolin Prior to Fertilization

    PubMed Central

    Finkelstein, Maya; Etkovitz, Nir; Breitbart, Haim

    2010-01-01

    To acquire fertilization competence, spermatozoa should undergo several biochemical changes in the female reproductive tract, known as capacitation. The capacitated spermatozoon can interact with the egg zona pellucida resulting in the occurrence of the acrosome reaction, a process that allowed its penetration into the egg and fertilization. Sperm capacitation requires actin polymerization, whereas F-actin must disperse prior to the acrosome reaction. Here, we suggest that the actin-severing protein, gelsolin, is inactive during capacitation and is activated prior to the acrosome reaction. The release of bound gelsolin from phosphatidylinositol 4,5-bisphosphate (PIP2) by PBP10, a peptide containing the PIP2-binding domain of gelsolin, or by activation of phospholipase C, which hydrolyzes PIP2, caused rapid Ca2+-dependent F-actin depolymerization as well as enhanced acrosome reaction. Using immunoprecipitation assays, we showed that the tyrosine kinase SRC and gelsolin coimmunoprecipitate, and activating SRC by adding 8-bromo-cAMP (8-Br-cAMP) enhanced the amount of gelsolin in this precipitate. Moreover, 8-Br-cAMP enhanced tyrosine phosphorylation of gelsolin and its binding to PIP2(4,5), both of which inactivated gelsolin, allowing actin polymerization during capacitation. This actin polymerization was blocked by inhibiting the Src family kinases, suggesting that gelsolin is activated under these conditions. These results are further supported by our finding that PBP10 was unable to cause complete F-actin breakdown in the presence of 8-Br-cAMP or vanadate. In conclusion, inactivation of gelsolin during capacitation occurs by its binding to PIP2 and tyrosine phosphorylation by SRC. The release of gelsolin from PIP2 together with its dephosphorylation enables gelsolin activation, resulting in the acrosome reaction. PMID:20937821

  1. The relationship between sperm head retardance using polarized light microscopy and clinical outcomes.

    PubMed

    Vermey, Belinda G; Chapman, Michael G; Cooke, Simon; Kilani, Suha

    2015-01-01

    In human sperm head, birefringence can be seen under polarized light resulting from highly ordered structures within the acrosome and nucleus. Selecting sperm with partial head birefringence improves success of clinical pregnancies in patients with severe male factor infertility. The aim of this study was to establish a range of retardance in sperm heads using polarized light microscopy to select an optimum sperm for intracytoplasmic sperm injection (ICSI). Sperm heads of 63 couples undergoing ICSI in women aged 38 years or younger were imaged at the time of ICSI and later analysed for retardance blinded to embryo and cycle outcomes. Sperm head retardance was similar irrespective of whether fertilization occurred. Quality of embryos on day 3 and day 5 were higher when sperm were selected with head retardance ranging from 0.56 nm or greater to 0.91 nm or less. Selection of sperm with head retardance ranging from 0.56 nm or greater to 0.91 nm or less was associated with higher clinical pregnancy rates (OR 3.74 95% CI 1.43 to 9.77). Optimum sperm for selection at the time of ICSI was with head retardance within the range 0.56 nm or greater to 0.91 nm or less. Copyright © 2014 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.

  2. Electron microscopic observation of the sagittal structure of Drosophila mature sperm.

    PubMed

    Yasuno, Yusaku; Yamamoto, Masa-Toshi

    2014-09-01

    Observation of sperm development and determination of their morphological characteristics are very important to the understanding of phylogenetic relationships and the study of sperm function during fertilization. Although ultrastructural studies of sperm development in the testes of the fruit fly Drosophila have been performed, there are few reports describing electron microscopic morphology of mature sperm, that is, those released from the testes to the seminal vesicles. Here, we present the first report of the sagittal organization of Drosophila sperm head and neck regions by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The head and tail structures of a mature sperm, for example, the acrosome, nucleus, and flagellum, were easy to distinguish by the morphological characteristics of the sperm surface by SEM. The morphological relationships between the surface and internal structures of mature sperm were confirmed by observing longitudinal sections with TEM. Our approach overcame the technical difficulties involved in sample preparation for electron microscopic observation of the Drosophila mature sperm head, and therefore, this study serves as an important foundation for future genetic dissection of sperm ultrastructure and function in male sterile mutants.

  3. Uterine flushings from women treated with levonorgestrel affect sperm functionality in vitro.

    PubMed

    Chirinos, Mayel; Durand, Marta; González-González, María Elena; Hernández-Silva, Gabriela; Maldonado-Rosas, Israel; Lopez, Pablo; Larrea, Fernando

    2017-09-08

    Levonorgestrel (LNG), a synthetic 19 nor-testosterone derivative, is widely used for emergency contraception. It is well known that LNG prevents ovulation only when given prior to the surge of serum luteinizing hormone (LH) during the periovulatory phase of the menstrual cycle. This observation suggests that LNG, given its contraceptive efficacy, has additional effects other than those affecting ovulation. In this study, we have evaluated the effects on human sperm functionality of uterine flushings (UF) obtained from women at day LH+1 of a control cycle (CTR-LH+1) and after receiving LNG (LNG-LH+1) two days before the surge of LH. Human sperm from normozoospermic donors were incubated with UF and protein tyrosine phosphorylation, sperm motility, acrosome reaction as well as zona pellucida (ZP) binding capacity were assessed. A significant decrease in total motility and tyrosine phosphorylation accompanied by an increase on spontaneous acrosome reaction was observed when sperm were incubated in the presence of LNG-LH+1. None of these effects were mimicked by purified glycodelin A (GdA). Moreover, the addition of UF obtained during the periovulatory phase from LNG-treated women or the presence of purified GdA significantly decreased sperm-ZP binding. The data were compatible with changes affecting sperm capacitation, motility and interaction with the ZP. These results may offer evidence on additional mechanisms of action of LNG as an emergency contraceptive.

  4. Zonadhesin is essential for species specificity of sperm adhesion to the egg zona pellucida.

    PubMed

    Tardif, Steve; Wilson, Michael D; Wagner, Rebecca; Hunt, Peter; Gertsenstein, Marina; Nagy, Andras; Lobe, Corrinne; Koop, Ben F; Hardy, Daniel M

    2010-08-06

    Interaction of rapidly evolving molecules imparts species specificity to sperm-egg recognition in marine invertebrates, but it is unclear whether comparable interactions occur during fertilization in any vertebrate species. In mammals, the sperm acrosomal protein zonadhesin is a rapidly evolving molecule with species-specific binding activity for the egg zona pellucida (ZP). Here we show using null mice produced by targeted disruption of Zan that zonadhesin confers species specificity to sperm-ZP adhesion. Sperm capacitation selectively exposed a partial von Willebrand D domain of mouse zonadhesin on the surface of living, motile cells. Antibodies to the exposed domain inhibited adhesion of wild-type spermatozoa to the mouse ZP but did not inhibit adhesion of spermatozoa lacking zonadhesin. Zan(-/-) males were fertile, and their spermatozoa readily fertilized mouse eggs in vitro. Remarkably, however, loss of zonadhesin increased adhesion of mouse spermatozoa to pig, cow, and rabbit ZP but not mouse ZP. We conclude that zonadhesin mediates species-specific ZP adhesion, and Zan(-/-) males are fertile because their spermatozoa retain adhesion capability that is not species-specific. Mammalian sperm-ZP adhesion is therefore molecularly robust, and species-specific egg recognition by a protein in the sperm acrosome is conserved between invertebrates and vertebrates, even though the adhesion molecules themselves are unrelated.

  5. A strategy for improvement of postthaw quality of bison sperm.

    PubMed

    Hussain, S A; Lessard, C; Anzar, M

    2013-01-01

    The objective was to improve the postthaw quality of bison semen using zwitterion (ZI)-based extenders, glycerol addition at a lower temperature (4 °C), adding reduced glutathione (GSH) in extender, or treating bison sperm with cholesterol-loaded cyclodextrin (CLC) before freezing. Postthaw sperm motility and structural characteristics were analyzed using a computer-assisted sperm analyzer and flow cytometer respectively, at 0 and 3 hours postthaw incubation at 37 °C. In experiment 1, each ejaculate (N = 11) was diluted in Triladyl extender (control) or in ZI extenders (Tes-Tris or HEPES-Tris). In addition, glycerol in semen was added either at 37 °C or 4 °C before cryopreservation. Extenders had no significant effect on postthaw sperm motilities at 0 hour. However, sperm velocity parameters were higher (P < 0.05) in ZI extenders than in Triladyl. Sperm population with intact plasma membrane (IPM) and acrosomes (IACR) were higher in Triladyl than in ZI extenders (P < 0.05). Postthaw sperm total and progressive motilities, average path velocity, straight-line velocity, IPM, and IPM-IACR did not improve with the addition of glycerol at 4 °C. In experiment 2, semen was diluted (50 × 10(6) sperm per mL) in Triladyl extender containing 0 (control), 0.5, 1.0, or 2.0 mM GSH (an antioxidant) at 37 °C. Postthaw sperm motility and structural characteristics at 0 hour and percentage declined after 3 hour incubation, but did not differ because of GSH in the extender (P > 0.05). In experiment 3, fresh bison sperm (100 × 10(6) sperm in 1 mL) were pretreated with 0, 1, 2, or 3 mg/mL of CLC at 22 °C for 15 minutes and diluted to 50 × 10(6) sperm per mL in Tris-citric acid-egg yolk-glycerol extender before cryopreservation. The CLC pretreated sperm had higher (P < 0.05) postthaw total and progressive motilities, IPM, and IACR at 0 hour and less percentage of decline in these characteristics after 3 hour postthaw incubation. In conclusion, zwitterion extenders (Tes

  6. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities linked to chromatin condensation failure.

    PubMed

    Boitrelle, F; Albert, M; Petit, J-M; Ferfouri, F; Wainer, R; Bergere, M; Bailly, M; Vialard, F; Selva, J

    2013-08-01

    Since an embryo's ability to grow to the blastocyst stage and implant can be improved by selection of a normal spermatozoon with a vacuole-free head, this study set out to determine the nature of small sperm vacuoles observed under high magnification (>×6300). For 15 infertile men with various sperm profiles, high-magnification microscopy was used to select motile, morphometrically normal spermatozoa with no vacuoles (n=450) or more than two small vacuoles (each of which occupied less than 4% of the head's area; n=450). Spermatozoa acrosome reaction status and degree of chromatin condensation were analysed. Three-dimensional deconvolution microscopy was used to accurately image the nucleus and acrosome at all depths in all spermatozoa. In all 450 spermatozoa with small vacuoles, the latter were seen to be abnormal, DNA-free nuclear concavities. Spermatozoa with small vacuoles were significantly more likely than vacuole-free spermatozoa to have noncondensed chromatin (39.8% versus 9.3%, respectively; P<0.0001). There was no significant difference between the two groups of spermatozoa in terms of acrosome reaction status. No association between chromatin condensation and acrosome reaction status was observed. Small human sperm vacuoles observed under high magnification are pocket-like nuclear concavities related to failure of chromatin condensation.

  7. Effects of genetic captive-breeding protocols on sperm quality and fertility in the white-footed mouse.

    PubMed

    Malo, Aurelio F; Martinez-Pastor, Felipe; Alaks, Glen; Dubach, Jean; Lacy, Robert C

    2010-10-01

    Mice (Peromyscus leucopus noveboracensis) from a captive-breeding program were used to test the effects of three genetic breeding protocols (minimizing mean kinship [MK], random breeding, and selection for docility [DOC]) and inbreeding levels on sperm traits and fertility. Earlier, in generation 8, one DOC replicate went extinct because of poor reproductive success. By generation 10, spermatozoa from DOC mice had more acrosome and midpiece abnormalities, which were shown to be strong determinants of fertility, as well as lower sperm production and resistance to osmotic stress. In addition, determinants of fertility, including male and female components, were assessed in a comprehensive manner. Results showed that the probability (P) of siring litters is determined by sperm number, sperm viability, and midpiece and acrosome abnormalities; that the P of siring one versus two litters is determined by tail abnormalities; and that the total number of offspring is influenced by female size and proportion of normal sperm, showing the relative importance of different sperm traits on fertility. On average, males with 20% normal sperm sired one pup per litter, and males with 70% normal sperm sired eight pups per litter. Interestingly, the proportion of normal sperm was affected by docility but not by relatively low inbreeding. However, inbreeding depression in sperm motility was detected. In the MK group, inbreeding depression not only affected sperm motility but also fertility: An increase in the coefficient of inbreeding (f) of 0.03 reduced sperm motility by 30% and translated into an offspring reduction of three pups in second litters. A genetic load of 48 fecundity equivalents was calculated.

  8. The effects on boar sperm quality of dietary supplementation with omega-3 polyunsaturated fatty acids differ among porcine breeds.

    PubMed

    Yeste, Marc; Barrera, Xavier; Coll, David; Bonet, Sergi

    2011-07-01

    The present study was undertaken to shed light on the relationship between boar sperm quality and dietary supplementation with omega-3 polyunsaturated fatty acids, which has been reported inconsistently in the literature. With this aim, such effects were evaluated and compared among three different porcine breeds: Duroc, Large-White, and Pietrain. Animals were randomly separated into two groups and fed either with a control diet or with a diet supplemented with omega-3. Sperm quality of these boar (ejaculate volume, sperm concentration, sperm viability, acrosome and mitochondrial sheath integrity, sperm motility, sperm morphology, and osmotic resistance of spermatozoa) was assessed every week for a 26-week period. Supplementing boar's diet with omega-3 did not affect ejaculate volume, sperm concentration, sperm motility, sperm viability, and acrosome and mitochondrial sheath integrity. In contrast, supplemented diet positively affected both sperm morphology in Large-White and Pietrain breeds and the osmotic resistance of Pietrain spermatozoa. No effects were seen for the same sperm parameters in Duroc breed. These breed-differences in boar fed with the supplemented diet could explain the contradictions in literature and might be related with differences in the composition of plasma membrane among breeds reported by other authors. Because no harmful effects were observed in the three evaluated breeds, but positive effects in Large-White and Pietrain boar, we can conclude that omega-3 fatty acids may be added to boar's diet at the levels used in this study to improve their sperm quality. More research is, however, needed to determine how these fatty acids differently affect the morphology and the osmotic resistance of the spermatozoa in these breeds.

  9. [Effects of some extenders and monoamines on sperm cryopreservation in tree shrews (Tupaia belangeri)].

    PubMed

    Ping, Shu-Huang; Wang, Cai-Yun; Tang, Wen-Ru; Luo, Ying; Yang, Shi-Hua

    2012-02-01

    The tree shrew may be an important experimental animal for disease models in humans. The effects of some extenders and momamines on sperm cryopreservation will provide helpful data for experimentation of strains and conservation of genetic resources in tree shrews. Epididymal sperm were surgically harvested from male tree shrews captured around Kunming, China and sperm motility, acrosome integrity and fertility were assessed during cryopreservation. In Experiment 1 eight extenders (TTE, TCG, TCF, TTG, BWW, BTS, DM, and SR) supplemented with 0.4 mol/L DMSO were used to dilute the sperm: only TTE, DM and SR showed no differences in motility and acrosome integrity compared to fresh controls after equilibration. After freezing and thawing, sperm in any extender showed lower motility than fresh control and sperm in DM showed higher motility than other groups. However, BWW produced the lowest motility. For acrosome integrity, TTE and DM showed higher than BWW, BTS and SR after equilibration. The parameter in DM was higher than other groups (except TTE) after thawing. In Experiment 2 four penetrating cryoprotectant agents (CPA) [dimethyl-formamide (DF), formamide (F), dimethylacetamide (DA), and acetamide (A)] at 0.2 mol/L, 0.4 mol/L, 0.8 mol/L, and 1.2 mol/L, respectively were added to the DM extender. Motility showed no difference among CPA groups and non-CPA group (control) after equilibration, but all thawed sperm showed lower values in motility and acrosome integrity than pre-freezing groups. However, sperm in 0.8 mol/L DF and 0.4 mol/L DMSO showed higher values in both parameters than that in other CPA groups (P>0.05). In Experiment 3 the fertilization rate of oocytes inseminated with 0.4mol/L DMSO (50%) were higher than that with 0.8mol/L DF (16%). In conclusion, non-ion extenders supplemented with egg yolk may be better for sperm cryopreservation in tree shrews and cryoprotectant effects of monoamines agents should be further studied in this species.

  10. Cryopreservation with α-tocopherol and Sephadex filtration improved the quality of boar sperm.

    PubMed

    Satorre, M M; Breininger, E; Beconi, M T

    2012-10-15

    The objectives were to evaluate postthaw sperm quality and the response to an inducer of in vitro sperm capacitation in boar sperm, cryopreserved with (T) or without (C) α-tocopherol. Boar sperm frozen in 0.2-mL pellets were thawed and washed (W) or selected by three methods: Percoll discontinuous gradient (PS) or Sephadex (Sigma-Aldrich, St. Louis, MO, USA) (neutral [S] or with ion exchange [S+IO] columns). All separation methods enhanced sperm motility, plasma membrane integrity, and functionality and acrosome integrity for both C and T samples (P < 0.05). The best results were obtained with S and ionic Sephadex column. There was a decrease (P < 0.05) in capacitation-like changes in C samples separated with Sephadex (W: 19 ± 0.9%, PS: 22 ± 2.5%, S: 17 ± 1.2%, and S+IO: 17 ± 2.0%). Cryopreservation with α-tocopherol decreased (P < 0.05) the percentage of cryocapacitated sperm (W: 14 ± 0.7%, PS: 14 ± 1.0%, S: 13 ± 1.0%, and S+IO: 14 ± 0.9%) compared with C samples, without differences among selection techniques. Freezing with α-tocopherol and subsequent selection decreased lipid peroxidation (W: 20.79 ± 2.64 nmol thiobarbituric acid reactive substances (TBARS)/10(8) sperm; PS: 13.15 ± 2.39 nmol TBARS/10(8) sperm; S: 13.20 ± 2.18 nmol TBARS/10(8) sperm, and S+IO: 13.62 ± 2.76 nmol TBARS/10(8) sperm), with respect to washed and selected C samples (W: 37.69 ± 5.34 nmol TBARS/10(8) sperm, PS: 25.61 ± 5.85 nmol TBARS/10(8) sperm, S: 19.16 ± 3.28 nmol TBARS/10(8) sperm, and S+IO: 22.16 ± 6.09 nmol TBARS/10(8) sperm). In vitro capacitation levels were significantly higher for neutral Sephadex-selected T samples in comparison with C and unselected samples. These results were confirmed with a follicular fluid-induced acrosome reaction. In conclusion, cryopreserved sperm with α-tocopherol and subsequent Sephadex selection, improved postthaw quality and functionality of boar sperm, which could be useful for assisted reproductive techniques. Copyright

  11. Quality of canine spermatozoa retrieved by percutaneous epididymal sperm aspiration.

    PubMed

    Varesi, S; Vernocchi, V; Faustini, M; Luvoni, G C

    2013-02-01

    To investigate the feasibility of percutaneous epididymal sperm aspiration in dogs and whether it might provide a population of epididymal spermatozoa similar to the population that can be obtained by processing isolated epididymis caudae. Concentration and total sperm number, motility, morphology and acrosomal integrity of spermatozoa retrieved by percutaneous epididymal sperm aspiration, in vitro aspiration and mincing of the cauda of the epididymis were compared. Percutaneous epididymal sperm aspiration is a feasible procedure to retrieve a population of spermatozoa in dogs. Quality is similar to that of spermatozoa collected in vitro, although a wide variation amongst animals was observed. In case of ejaculation failure due to pathological conditions in dogs, the collection of spermatozoa from the cauda of the epididymis could be an option for providing gametes for assisted reproductive technologies. Percutaneous epididymal sperm aspiration can be used in dogs with compromised reproductive performance, in which orchiectomy cannot be performed for medical or owner reasons. Further studies aimed to investigate whether the percutaneous epididymal sperm aspiration technique might be feasible for repeated semen collection and to accurately evaluate side effects are required. © 2013 British Small Animal Veterinary Association.

  12. Biochemical and microscopic analysis of sperm in copper deficient mice

    SciTech Connect

    Everett, J.; Jackson, P.; Allison, S.

    1986-03-01

    The Mottle Brindle Mouse Syndrome is a disease in mice which mimics Menkes Syndrome in humans. Treatment of affected male mice has led to varying survival rates in mice and few attempts have led to the development of virile male offsprings in mice and none in humans. In this study the authors examined sperm produced by Brindle mice in an attempt to ascertain reasons for the observed failure of the Brindle mice to reproduce. Microscopic analysis revealed that sperm counts in these mice are higher than sperm counts of the C57/BL or the C57/6J (normal) mice. Microscopically, sperm from Brindle mice showed changes in the acrosomal and flagellum regions. Motility of these sperm were 10% to 50% that of sperm from normal mice. Biochemically, cytochrome oxidase activity was 10% to 50% of the activity seen in normal mice. Hexokinase activity and pyruvate dehydrogenase activity was equal to that observed in normal mice. These observations suggest that infertility in Brindle male mice is due to an impairment of testicular copper transport which leads to a decline in copper dependent processes.

  13. Chain Reacting System

    NASA Astrophysics Data System (ADS)

    Fermi, Enrico; Leverett, Miles C.

    presence in the system of high temperatures and intense neutron densities causes an acceleration of any normal rate of corrosion, resulting in the physical deterioration of the uranium in the system. It is essential, then, that the circulating medium be of such a character as not to destroy the uranium bodies in the system. In the present case, the cooling medium is gaseous helium circulating in the active regions of the reactor, which has the advantage of minimizing the possible corrosion of the fissile material, since it is an inert gas, and the absorption of neutrons. However, other possible choices, affecting the determination of the multiplication factor, for the coolant gas (such as air, oxigen or water vapor) are discussed as well in terms of their "danger coefficients", defined in terms of the ratio of the weight of impurity per unit mass of uranium and based on the cross section for absorption of thermal neutrons of the various elements [Fermi (1942a)]. The discussion of some methods of cooling chain reacting piles was initiated in [Fermi (1942g)], but no reference published paper exists of the material presented here.

  14. Sperm storage in the oviduct of the American alligator.

    PubMed

    Gist, Daniel H; Bagwill, April; Lance, Valentine; Sever, David M; Elsey, Ruth M

    2008-12-01

    Oviducts of the American alligator (Alligator mississippiensis) were examined histologically for the presence of stored sperm. Two regions containing sperm were identified, one at the junction of the posterior uterus and the vagina (UVJ) and the other at the junction of the tube and isthmus (TIJ). In these areas, sperm were found in the lumina of oviductal glands. The glands in these areas of the oviduct are diffuse and shallow and appear to allow better access to sperm than glands located elsewhere. Histochemically, the glands of the UVJ reacted weakly for carbohydrates and proteins, whereas those of the TIJ reacted strongly for these same two components, secretions of which are associated with sperm storage structures in other reptiles. Sperm were not in contact with the glandular epithelium, and glands at the UVJ contained more sperm than those at the TIJ. Oviductal sperm storage was observed not only in recently mated females but in all females possessing uterine eggs as well as all females known to be associated with a nest. We conclude that female alligators are capable of storing sperm in their oviductal glands, but not from one year to the next. (c) 2008 Wiley-Liss, Inc.

  15. Molecular identification of Ca2+ channels in human sperm.

    PubMed

    Park, Jin-Yong; Ahn, Hyun-Jong; Gu, Jung-Gyu; Lee, Keun-Ho; Kim, Jin-Sung; Kang, Ho-Won; Lee, Jung-Ha

    2003-08-31

    The acrosome reaction is a Ca(2+)-dependent exocytotic process that is a prerequisite step for fertilization. External calcium entry through voltage-activated Ca(2+) channels is known to be essential in inducing the acrosome reaction of mammalian spermatozoa. Due to their complex geometry, however, electrophysiological identification of sperm Ca(2+) channels has been limited. Here we identified Ca(2+) channel mRNAs expressed in motile human sperm using RT-PCR and their levels were compared using RNase protection assays. L-type, non- L-type, and T-type Ca(2+) channel mRNAs were detected by RT-PCR using degenerate primers. Cloning and sequencing of the PCR products revealed alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H sequences. RT-PCR using specific primers repeatedly detected alpha1B, alpha1C, alpha1E, alpha1G, and alpha1H mRNAs, and additionally alpha1I mRNA. But alpha1A and alpha1D messages were not detected. Relative expression levels of the detected Ca(2+) channel subtypes were compared by RNase protection assays. The abundance of detected mRNA messages was in the following order: alpha1H alpha1G alpha1E alpha1B alpha1C alpha1I. These findings indicated that human motile sperm express multiple voltage-activated Ca(2+) channel RNAs among which T-type and non-L-type channel messages are likely to be predominantly expressed. Based on their relative expression levels, we propose that not only T-type but also non-L-type calcium channels may be major gates for the external calcium influx, required for the acrosome reaction.

  16. Effect of colloid (Androcoll-Bear, Percoll, and PureSperm) selection on the freezability of brown bear (Ursus arctos) sperm.

    PubMed

    Álvarez-Rodríguez, M; Álvarez, M; Anel-López, L; López-Urueña, E; Manrique, P; Borragán, S; Morrell, J M; de Paz, P; Anel, L

    2016-04-01

    The development of a species-specific conservation protocol that involves artificial insemination with frozen semen needs to validate an effective methodology for freezing semen. Colloid centrifugation has been suggested and widely applied as an effective tool for selecting animal spermatozoa for artificial breeding. The objective of the present study was to compare different methods of centrifugation, single layer using Androcoll-Bear and Percoll and double layer using PureSperm 100 (in two different discontinuous gradients 40%-80% and 45%-90%), for the selection of fresh brown bear sperm samples. In the before freezing group, all selected samples showed a higher progressive motility and viability (except Percoll for motility 43.0 ± 5.3 [P < 0.05]); all colloids except PureSperm 45/90% rendered samples with fewer damaged acrosomes. In the after thawing group, all tested centrifugation colloids showed a good capacity to decrease the number of damaged acrosomes. Furthermore, PureSperm treatment (45/90%) resulted in an increase in apoptotic-like changes not only immediately after thawing but also after the incubation test, leading us to suggest that this gradient could induce some kind of deleterious effects on the sperm samples. On the other hand, PureSperm treatment (40/80%) yielded a quality preservation capacity similar to Androcoll-Bear in number of damaged acrosomes, different relative to the control (control, 5.3 ± 0.6; PureSperm 80, 2.0 ± 0.3; Androcoll, 2.1 ± 0.9 [P < 0.05]) but a decrease in the number of viable spermatozoa recovered after thawing relative to the control (control, 21.2 ± 3.1; PureSperm 80, 13.7 ± 2.7 [P < 0.05]). In conclusion, Androcoll-Bear constitutes a useful tool for handling of brown bear ejaculates owing to its simple handling and procedure with a reliable sperm selection and freezability. This colloid yielded an improvement in several sperm parameters in brown bear frozen-thawed semen; the selected spermatozoa of fresh samples

  17. Porcine sperm vitrification II: Spheres method.

    PubMed

    Arraztoa, C C; Miragaya, M H; Chaves, M G; Trasorras, V L; Gambarotta, M C; Neild, D M

    2016-11-10

    Owing to current problems in boar sperm cryopreservation, this study proposes to evaluate vitrification in spheres as an alternative cryopreservation procedure, comparing the use or not of permeable cryoprotectants and two warming methods. Extended (n = 3; r = 4) and raw (n = 5; r = 2) porcine spermatozoa were diluted in media, in the absence or presence of either 4% dimethylformamide or 4% glycerol, to a final concentration of 5 × 10(6)  spermatozoa/ml and vitrified using the spheres method. Two warming procedures were evaluated: a rapid method (30 s at 37°C) and an ultrarapid method (7 s at 75°C, followed by 30 s at 37°C). Percentages of total motility (phase contrast), membrane function (hypo-osmotic swelling test), acrosome integrity (phase contrast), sperm viability (6-carboxyfluorescein diacetate and propidium iodide stain), chromatin condensation (toluidine blue stain) and chromatin susceptibility to acid denaturation (acridine orange stain) were evaluated in the samples before and after vitrification. Results, analysed using Friedman's test, suggest that rapid warming of raw porcine spermatozoa vitrified without permeable cryoprotectants may preserve DNA condensation and integrity better than the other processing methods studied in this work. Hence, porcine sperm vitrification using spheres could be used to produce embryos with ICSI to further validate this method.

  18. Induced lipid peroxidation in ram sperm: semen profile, DNA fragmentation and antioxidant status.

    PubMed

    Hamilton, Thais Rose dos Santos; de Castro, Letícia Signori; Delgado, Juliana de Carvalho; de Assis, Patrícia Monken; Siqueira, Adriano Felipe Perez; Mendes, Camilla Mota; Goissis, Marcelo Demarchi; Muiño-Blanco, Teresa; Cebrián-Pérez, José Álvaro; Nichi, Marcílio; Visintin, José Antonio; D'Ávila Assumpção, Mayra Elena Ortiz

    2016-04-01

    Action of reactive oxygen species, protamination failures and apoptosis are considered the most important etiologies of sperm DNA fragmentation. This study evaluated the effects of induced lipid peroxidation susceptibility on native semen profile and identified the mechanisms involved in sperm DNA fragmentation and testicular antioxidant defense on Santa Ines ram sperm samples. Semen was collected from 12 adult rams (Ovis aries) performed weekly over a 9-week period. Sperm analysis (motility, mass motility, abnormalities, membrane and acrosome status, mitochondrial potential, DNA fragmentation, lipid peroxidation and intracellular free radicals production); protamine deficiency; PRM1, TNP1 and TNP2 gene expression; and determination of glutathione peroxidase (GPx), glutathione reductase, catalase (CAT) and superoxide dismutase activity and immunodetection in seminal plasma were performed. Samples were distributed into four groups according to the sperm susceptibility to lipid peroxidation after induction with ascorbate and ferrous sulfate (low, medium, high and very high). The results were analyzed by GLM test and post hoc least significant difference. We observed an increase in native GPx activity and CAT immunodetection in groups with high susceptibility to induced lipid peroxidation. We also found an increase in total sperm defects, acrosome and membrane damages in the group with the highest susceptibility to induced lipid peroxidation. Additionally, the low mitochondrial membrane potential, susceptible to chromatin fragmentation and the PRM1 mRNA were increased in the group showing higher susceptibility to lipid peroxidation. Ram sperm susceptibility to lipid peroxidation may compromise sperm quality and interfere with the oxidative homeostasis by oxidative stress, which may be the main cause of chromatin damage in ram sperm. © 2016 Society for Reproduction and Fertility.

  19. Effects of different concentrations of Pseudomonas aeruginosa on boar sperm quality.

    PubMed

    Sepúlveda, Lilian; Bussalleu, Eva; Yeste, Marc; Bonet, Sergi

    2014-11-30

    Bacteriospermia in boar ejaculates is a frequent finding that compromises the sperm quality and, consequently, causes economic losses in swine industry. The present study sought to evaluate the effect of different concentrations of Pseudomonas aeruginosa on boar sperm quality over a storing period of 11 days at 15-17 ° C. Ten commercial seminal doses coming from post-pubertal and healthy boars were artificially inoculated with different infective concentrations of P. aeruginosa, ranging from 2 × 10(8) to 2 × 10(4)cfu/mL. Negative controls were non-inoculated doses. Sperm quality, assessed as sperm motility (CASA), sperm viability, acrosome integrity and pH, as well as the bacterial growth, were checked after 0, 1, 2, 4, 7, 9 and 11 days of storage at 15-17 ° C. Results obtained showed significant decreases in the percentages of total and progressive sperm motility, sperm viability and acrosome integrity in the greatest infective concentrations (2 × 10(7) and 2 × 10(8)cfu/mL), when compared to the negative control. In contrast, there was no effect on seminal pH throughout the experiment. Results indicate the presence of P. aeruginosa in boar semen, apart from being a potential source for the spread of infectious diseases and harmful impact on sows, negatively affects the longevity and fertilizing ability of boar sperm when present in high concentrations. Thus, P. aeruginosa causes deleterious effects on boar sperm quality during liquid storage at 15-17 ° C, thus strict hygienic measures must be implemented in boar studs to minimize bacterial concentration of semen doses.

  20. Oocyte Activation and Fertilisation: Crucial Contributors from the Sperm and Oocyte.

    PubMed

    Yeste, Marc; Jones, Celine; Amdani, Siti Nornadhirah; Coward, Kevin

    2017-01-01

    This chapter intends to summarise the importance of sperm- and oocyte-derived factors in the processes of sperm-oocyte binding and oocyte activation. First, we describe the initial interaction between sperm and the zona pellucida, with particular regard to acrosome exocytosis. We then describe how sperm and oocyte membranes fuse, with special reference to the discovery of the sperm protein IZUMO1 and its interaction with the oocyte membrane receptor JUNO. We then focus specifically upon oocyte activation, the fundamental process by which the oocyte is alleviated from metaphase II arrest by a sperm-soluble factor. The identity of this sperm factor has been the source of much debate recently, although mounting evidence, from several different laboratories, provides strong support for phospholipase C ζ (PLCζ), a sperm-specific phospholipase. Herein, we discuss the evidence in support of PLCζ and evaluate the potential role of other candidate proteins, such as post-acrosomal WW-binding domain protein (PAWP/WBP2NL). Since the cascade of downstream events triggered by the sperm-borne oocyte activation factor heavily relies upon specialised cellular machinery within the oocyte, we also discuss the critical role of oocyte-borne factors, such as the inositol trisphosphate receptor (IP3R), protein kinase C (PKC), store-operated calcium entry (SOCE) and calcium/calmodulin-dependent protein kinase II (CaMKII), during the process of oocyte activation. In order to place the implications of these various factors and processes into a clinical context, we proceed to describe their potential association with oocyte activation failure and discuss how clinical techniques such as the in vitro maturation of oocytes may affect oocyte activation ability. Finally, we contemplate the role of artificial oocyte activating agents in the clinical rescue of oocyte activation deficiency and discuss options for more endogenous alternatives.

  1. Morphology and function of the reproductive tract of the spider crab Libinia spinosa (Crustacea, Brachyura, Majoidea): pattern of sperm storage

    NASA Astrophysics Data System (ADS)

    Sal Moyano, M. P.; Gavio, M. A.; Cuartas, E. I.

    2010-09-01

    Morphology and function of the male reproductive tract, female spermatheca and patterns of sperm storage were assessed in the crab Libinia spinosa using histological methods. Testes are characterized by the presence of peripheral spermatogonia and different sequences of sperm maturity. Spermatophores begin to be packed in the last portion. The vas deferens consists of three sections: anterior, with undeveloped spermatophores and free sperm; median, with well-developed spermatophores; and posterior with granular secretions. Female spermathecae are of the ventral type, with a velum separating dorsal and ventral chambers. Live individuals were kept in the laboratory and arranged in pairs. An experiment was conducted toward the end of the reproductive season, in which males with the right gonopod excised were placed with receptive females. After mating, females were killed and the spermathecae dissected for histological study and observation of the pattern of sperm storage. Spermatozoa were found forming discrete sperm packages. New ejaculates can fill the entire spermatheca or be restricted to the ventral chamber; sperm are rounded, with a distinguishable acrosomal core. Old ejaculates are restricted to the dorsal chamber and are of irregular shape and larger size; an acrosomal core was not distinguishable. The secretions produced by the glandular epithelium of the dorsal chamber of the spermathecae are likely to have a role in the removal of dead sperm.

  2. Antibodies to spermatozoa. III. Responses in rabbits and guinea-pigs to immunization with guinea-pig sperm cells

    PubMed Central

    Hekman, Annemarie; Shulman, S.

    1971-01-01

    The antigens of guinea-pig sperm cells, of both the epididymal and ejaculated (or seminal) types, have been studied, using rabbit and guinea-pig antisera. Several antigens could be revealed by gel diffusion studies, using well-washed but non-ruptured sperm cells, indicating that intentional cell breaking is not essential for demonstrating the antigens. This release of soluble antigen was followed as a function of time and temperature, both as total protein in supernatants and in increasing strength of precipitation. With rabbit antiserum, epididymal sperm showed two antigens, that were also demonstrated in epididymal and testicular extract and in seminal sperm. These other materials revealed additional antigens with these antisera. Immunofluorescent staining was limited to the acrosomes. With guinea-pig antibodies, no precipitating antigen that was characteristic of sperm could be seen. These antisera showed immunofluorescent staining of the acrosomes. The staining could be distinguished, in terms of thermostability, from the staining produced by normal serum. No evidence was found for the occurrence of any sperm-coating antigens in the guinea-pig, especially since both antiseminal plasma and antivesicular fluid antisera failed to give immunofluorescent staining of the sperm cells. ImagesFig. 1Fig. 3Fig. 4Fig. 5Fig. 6 PMID:4998924

  3. K+ and Cl− Channels and Transporters in Sperm Function

    PubMed Central

    Santi, C.M.; Orta, G.; Salkoff, L.

    2013-01-01

    To succeed in fertilization, spermatozoa must decode environmental cues which require a set of ion channels. Recent findings have revealed that K+ and Cl− channels participate in some of the main sperm functions. This work reviews the evidence indicating the involvement of K+ and Cl− channels in motility, maturation, and the acrosome reaction, and the advancement in identifying their molecular identity and modes of regulation. Improving our insight on how these channels operate will strengthen our ability to surmount some infertility problems, improve animal breeding, preserve biodiversity, and develop selective and secure male contraceptives. PMID:23287041

  4. Flying-fox (Pteropus spp.) sperm membrane fatty acid composition, its relationship to cold shock injury and implications for cryopreservation success.

    PubMed

    Melville, D F; Johnston, S D; Miller, R R

    2012-12-01

    The very large acrosome of Pteropus species spermatozoa is prone to damage during cooling procedures. Cryogenic succuss has been linked to membrane composition, therefore the lipid composition of five Pteropus species sperm acrosomal and plasma membranes were investigated to provide insight into reasons for cold shock susceptibility. Rapid chilling and re-warming of spermatozoa from three Pteropus species resulted in a decrease (P<0.05) in acrosomal integrity. Biochemical analysis of lipids revealed that stearic acid (18:0) was the predominant saturated fatty acid and oleic acid (18:1, n-9) the predominant unsaturated fatty acid in both acrosomal and plasma membranes. Linolenic acid (18:3, n-3) was only detected in plasma membranes of Pteropus hypomelanus and was detected in acrosomal membranes of all Pteropus spp. studied (except Pteropus giganteus). Although detected in both plasma and acrosomal membranes of Pteropus vampyrus, docosahexaenoic acid (22:6) was not detected at all in Pteropus poliocephalus, only in trace levels in the acrosomal and plasma membranes of P. giganteus and P. hypomelanus and not in acrosomal membranes of Pteropus rodricensis. No difference was seen in the levels of polyunsaturated fatty acids (PUFAs) within plasma membranes, however PUFAs were lower (P<0.05) in acrosomal membranes of P. giganteus compared with P. vampyrus. Pteropus spp. spermatozoa have a very low ratio of unsaturated/saturated membrane fatty acids (<0.5). Membranes containing more PUFAs are more fluid, so the use of cryogenic media which improves membrane fluidity should improve Pteropus spp. spermatozoal viability post-thaw. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation

    PubMed Central

    Saucedo, Lucía; Buffa, Gabriela N.; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J.

    2015-01-01

    Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility. PMID:25970615

  6. Fibroblast Growth Factor Receptors (FGFRs) in Human Sperm: Expression, Functionality and Involvement in Motility Regulation.

    PubMed

    Saucedo, Lucía; Buffa, Gabriela N; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrian; Munuce, María J; Vazquez-Levin, Mónica H; Marín-Briggiler, Clara

    2015-01-01

    Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.

  7. [Influence of Ureaplasma urealyticum infection on the sperm-egg binding associated molecule, sulfogalactosylglycerolipid].

    PubMed

    Wu, Tingting; Lu, Meige; Hu, Yanqin; Guo, Qiangsu; Xu, Chen

    2004-09-01

    To study the influence of Ureaplasma urealyticum (Uu) infection on the sperm-egg binding associated molecule, sulfogalactosylglycerolipid (SGG). Epididymal sperm was collected from adult mice. The sperm suspension was randomly divided into 4 groups: Uu group (coincubated with Uu suspension), medium group (coincubated with Uu medium), normal group and PRS group. The indirect immunofluorescence technique was used to localize SGG on the sperm membrane and to observe the influence of Uu on SGG. In the epididymal sperm, SGG was localized to the head plasma membrane overlaying the acrosomal region. The SGG-positive rate of the sperm coincubated with Uu medium was 82.0%, while that of the sperm coincubated with Uu suspension was reduced to 39.0% (P = 0.001). Uu can adhere to the sperm surface. SGG might be a membrane receptor on the sperm surface for Uu infection of the mammalian male genital tract. The blockage of SGG by Uu might be one of the molecular mechanisms correlative to male infertility induced by Uu infection.

  8. The nature of human sperm head vacuoles: a systematic literature review.

    PubMed

    Boitrelle, Florence; Guthauser, Bruno; Alter, Laura; Bailly, Marc; Wainer, Robert; Vialard, François; Albert, Martine; Selva, Jacqueline

    2013-01-01

    Motile sperm organelle morphology examination (MSOME) involves the use of differential interference contrast microscopy (also called Nomarski contrast) at high magnification (at least 6300x) to improve the observation of live human spermatozoa. In fact, this technique evidences sperm head vacuoles that are not necessarily seen at lower magnifications - particularly if the vacuoles are small (i.e. occupying <4% of the sperm head's area). However, a decade after MSOME's introduction, it is still not clear whether sperm head vacuoles are nuclear, acrosomal and/or membrane-related in nature. In an attempt to clarify this debate, we performed a systematic literature review in accordance with the PRISMA guidelines. The PubMed database was searched from 2001 onwards with the terms "MSOME", "human sperm vacuoles", "high-magnification, sperm". Out of 180 search results, 21 relevant English-language publications on the nature of human sperm head vacuoles were finally selected and reviewed. Our review of the literature prompted us to conclude that sperm-head vacuoles are nuclear in nature and are related to chromatin condensation failure and (in some cases) sperm DNA damage.

  9. The molecular chaperone HSPA2 plays a key role in regulating the expression of sperm surface receptors that mediate sperm-egg recognition.

    PubMed

    Redgrove, Kate A; Nixon, Brett; Baker, Mark A; Hetherington, Louise; Baker, Gordon; Liu, De-Yi; Aitken, R John

    2012-01-01

    A common defect encountered in the spermatozoa of male infertility patients is an idiopathic failure of sperm-egg recognition. In order to resolve the molecular basis of this condition we have compared the proteomic profiles of spermatozoa exhibiting an impaired capacity for sperm-egg recognition with normal cells using label free mass spectrometry (MS)-based quantification. This analysis indicated that impaired sperm-zona binding was associated with reduced expression of the molecular chaperone, heat shock 70 kDa protein 2 (HSPA2), from the sperm proteome. Western blot analysis confirmed this observation in independent patients and demonstrated that the defect did not extend to other members of the HSP70 family. HSPA2 was present in the acrosomal domain of human spermatozoa as a major component of 5 large molecular mass complexes, the most dominant of which was found to contain HSPA2 in close association with just two other proteins, sperm adhesion molecule 1 (SPAM1) and arylsulfatase A (ARSA), both of which that have previously been implicated in sperm-egg interaction. The interaction between SPAM1, ARSA and HSPA2 in a multimeric complex mediating sperm-egg interaction, coupled with the complete failure of this process when HSPA2 is depleted in infertile patients, provides new insights into the mechanisms by which sperm function is impaired in cases of male infertility.

  10. Effects of very rapid versus vapor phase freezing on human sperm parameters.

    PubMed

    Darvishnia, Hamid; Lakpour, Niknam; Lahijani, Maryam Shams; Heidari-Vala, Hamed; Akhondi, Mohammad A; Zeraati, Hojjat; Sadeghi, Mohammad Reza

    2013-12-01

    The aim of the present study was to compare the effects of two freezing methods, vapor phase and very rapid freezing, with and without cryoprotectant on semen parameters in men with normal semen criteria. Cryopreservation was done on semen samples from 31 men by two methods of vapor phase freezing and very rapid freezing, with and without Test Yolk buffered glycerol (TYBG) as cryoprotectant. The motility, viability, acrosome and DNA integrity were evaluated on fresh and post-thaw samples. Post-thaw sperm progressive motility was significantly higher in the presence of TYBG in the vapor phase cryopreservation (%6.30 ± 3.74) compared with the very rapid freezing method (%2.2 ± 1.97 and %4.00 ± 2.42 in the presence and absence of TYBG, respectively). There was no significant difference in viability, acrosome status and DNA integrity between two methods in presence or absence of TYBG. The very rapid freezing method in the absence of TYBG showed better sperm motility but viability, acrosome and DNA integrity were similar to the presence of TYBG. The results show that cryopreservation of human spermatozoa together with seminal plasma by using vapor phase method is better than very rapid freezing method to preserve sperm progressive motility; however very rapid freezing method is quick and simple and do not require special cryoprotectant. It can be used for cryopreservation of small number of spermatozoa in IVF centers.

  11. Semen Quality and Sperm Function Loss by Hypercholesterolemic Diet Was Recovered by Addition of Olive Oil to Diet in Rabbit

    PubMed Central

    Romero, Aida A.; Funes, Abi K.; Cid-Barria, Macarena; Cabrillana, María E.; Monclus, María A.; Simón, Layla; Vicenti, Amanda E.; Fornés, Miguel W.

    2013-01-01

    Fat increment (0.05% cholesterol, chol) in standard diet promoted a significant increase in serum and sperm membrane chol, which ultimately altered membrane-coupled sperm specific functions: osmotic resistance, acrosomal reaction, and sperm capacitation in White New Zealand rabbits. These changes were also associated with a reduction in motility percentage and appearance of abnormal sperm morphology. The present study was carried out to evaluate the effect of dietary olive oil (OO, 7% v/w) administration to several male hypercholesterolemic rabbits (hypercholesterolemic rabbits, HCR) with altered fertility parameters. These HCR males were achieved by feeding normal rabbits with a high-fat diet (0.05% chol). HCR were associated with a modest non-significant increase in body weight (standard diet, 4.08±0.17 Kg, versus high-fat diet, 4.37±0.24 Kg). Hypercholesterolemic rabbits presented a marked decrease in semen volume, sperm cell count, and percentage of sperm motility, associated with a significant increase in sperm cell abnormalities. Moreover, sperm capacitation measured by the characteristic phosphorylated protein pattern in and induced acrosomal reaction were also altered suggesting sperm dysfunction. However, the administration of OO (for 16 weeks) to rabbits that were fed with 50% of the high-fat diet normalized serum chol. Curiously, OO supply succeeded to attenuate the seminal and sperm alterations observed in HCR group. Administration of OO alone did not cause any significant changes in above mentioned parameters. These data suggest that OO administration to HCR male rabbits recovers the loss of semen quality and sperm functionality. PMID:23326331

  12. Retained functional integrity of bull spermatozoa after double freezing and thawing using PureSperm density gradient centrifugation.

    PubMed

    Maxwell, W M C; Parrilla, I; Caballero, I; Garcia, E; Roca, J; Martinez, E A; Vazquez, J M; Rath, D

    2007-10-01

    The main aim of this study was to compare the motility and functional integrity of bull spermatozoa after single and double freezing and thawing. The viability and morphological integrity of spermatozoa selected by PureSperm density gradient centrifugation after cryopreservation of bovine semen in two commercial extenders (Experiment 1) and the function of bull spermatozoa before and after a second freezing and thawing assisted by PureSperm selection (Experiment 2) were examined. On average, 35.8 +/- 12.1% of sperm loaded onto the PureSperm density gradient were recovered after centrifugation. In Experiment 1, post-thaw motility and acrosome integrity were higher for spermatozoa frozen in Tris-egg yolk extender than in AndroMed, whether the assessments were made immediately after thawing [80.4 +/- 12.7 vs 47.6 +/- 19.0% motile and 78.8 +/- 8.3 vs 50.1 +/- 19.5% normal apical ridge (NAR), p < 0.05] or after preparation on the gradient (83.3 +/- 8.6 vs 69.4 +/- 15.9% motile and 89.5 +/- 7.2 vs 69.1 +/- 11.4% NAR, p < 0.05). For semen frozen in Tris-egg yolk extender, selection on the PureSperm gradient did not influence total motility but significantly improved the proportion of acrosome-intact spermatozoa. After the gradient, both the total motility and percentage of normal acrosomes increased for spermatozoa frozen in AndroMed (Minitüb Tiefenbach, Germany). In Experiment 2, there was no difference in sperm motility after the first and second freeze-thawing (82.9 +/- 12.7 vs 68.8 +/- 18.7%). However, the proportion of acrosome-intact spermatozoa was significantly improved by selection through the PureSperm gradient, whether measured by phase contrast microscopy (78.9 +/- 9.7 vs 90.4 +/- 4.0% NAR, p < 0.05) or flow cytometry (53.4 +/- 11.7 vs 76.3 +/- 6.0% viable acrosome-intact spermatozoa, p < 0.001). The improvement in the percentage of spermatozoa with normal acrosomes was maintained after resuspension in the cooling extender and cooling to 4 degrees C (88

  13. Evaluation of sperm quality snakes Erythrolamprus poecilogyrus sublineatus (Cope, 1860) (Serpentes, Dipsadidae).

    PubMed

    Silva, A C; Varela, A S; Cardoso, T F; Silva, E F; Loebmann, D; Corcini, C D

    2017-01-12

    Erythrolamprus poecilogyrus sublineatus (Cope, 1860), is a species widely distributed in the Pampa Domain, occurring in Rio Grande do Sul, Argentina and Uruguay, mainlyin the pampa region. In the coastal region of southern Brazil this is serpent is considered one of the most abundant. The purpose of the present study is to describe the techniques of sperm evaluation in vitro for E. poecilogyrus sublineatus in the coastal plain of Rio Grande do Sul, Brazil. After laparatomy the efferent vases were collected and the semen was diluted in 1ml Beltsville Thawing Solution. The characteristics of motility, membrane integrity, mitochondria, acrosome, DNA, cell viability and cellular functionality were evaluated. Fluorescent probes were used for the evaluation of sperm structure in epifluorescence microscope. With the techniques described, it was possible to identify intact and injured cells, enabling the determination of cell characteristics for the spring season (October and November). It was observed in the analyses that 80% of sperm cells were mobile and that 84.1 ± 8.0% of sperm membranes were intact. The standards found were of 48 ± 13.8% of intact acrosome, 73.6 ± 6.0 of perfect DNA and of 91.8 ± 4.0 of functional mitochondria. Thus, these values from the sperm analysis can be used as standards for the species Erythrolamprus poecilogyrus sublineatus.

  14. Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica).

    PubMed

    Sasanami, Tomohiro; Sugiura, Kenichi; Tokumoto, Toshinobu; Yoshizaki, Norio; Dohra, Hideo; Nishio, Shunsuke; Mizushima, Shusei; Hiyama, Gen; Matsuda, Tsukasa

    2012-10-01

    At the time of fertilization, the extracellular matrix surrounding avian oocytes, termed the perivitelline membrane (pvm), is hydrolyzed by a sperm-borne protease, although the actual protease that is responsible for the digestion of the pvm remains to be identified. Here, we show evidence that the ubiquitin-proteasome system is functional in the fertilization of Japanese quail. The activities for the induction of the acrosome reaction and binding to ZP3 as revealed by ligand blotting of purified serum ZP1 are similar to those of pvm ZP1. Western blot analysis of purified ZP1 and ZP3 by the use of the anti-ubiquitin antibody showed that only pvm ZP1 was reactive to the antibody. In vitro penetration assay of the sperm on the pvm indicated that fragments of ZP1 and intact ZP3 were released from the pvm. Western blot analysis using the anti-20S proteasome antibody and ultrastructural analysis showed that immunoreactive proteasome was localized in the acrosomal region of the sperm. Inclusion of specific proteasome inhibitor MG132 in the incubation mixture, or depletion of extracellular ATP by the addition of apyrase, efficiently suppressed the sperm perforation of the pvm. These results demonstrate for the first time that the sperm proteasome is important for fertilization in birds and that the extracellular ubiquitination of ZP1 might occur during its transport via blood circulation.

  15. Thawed human sperm quality is influenced by the volume of the cryopreserved specimen.

    PubMed

    Abush, Ayelet; Hauser, Ron; Paz, Gedalia; Kleiman, Sandra E; Lehavi, Ofer; Yavetz, Haim; Yogev, Leah

    2014-03-01

    To test the effect of sperm specimen volume in the freezing-thawing process on specimen quality. Experimental prospective study. Tertiary academic medical center. Fifty high-quality sperm donors donated ∼3 times each. Sperm samples were split into two aliquots and frozen in volumes of 0.25 mL and 0.5 mL. Semen analyses. Eight sperm quality parameters of thawed specimens. Thawed 0.5-mL specimens had a higher percentage of motility and viability, progressive motility concentration, percentage of cells with high mitochondrial membrane potential, and intact chromatin compared with 0.25-mL specimens. Although there were fewer cells with intact acrosomes in the 0.5-mL thawed samples, they had a similar ability to respond to ionophore by acrosome reaction as the 0.25-mL specimens. Both groups had similar percentages of cells with oxidative stress and numbers of cells that bound to the zona pellucida. The remaining air volume in the straw and freezing medium composition had a minimal effect on tested parameters. Better quality thawed human sperm was achieved after cryopreservation of high volumes compared with low volumes of specimens. Air volume in the straw had no influence on specimen quality. Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.

  16. The sperm ultrastructure and spermiogenesis of Tribolium castaneum (Coleoptera: Tenebrionidae) with evidence of cyst degeneration.

    PubMed

    Dias, Glenda; Lino-Neto, José; Mercati, David; Dallai, Romano

    2015-06-01

    Previous studies on the spermatogenesis of tenebrionid beetles showed the unusual formation of two antiparallel sperm bundles per cyst. In this work we reported this feature also in Tribolium castaneum using light and transmission electron microscopy. The sperm structure of T. castaneum, similar to other tenebrionids, consists of a three-layered acrosome, an elongated nucleus and a flagellum with a 9+9+2 axoneme, two accessory bodies and two asymmetric mitochondrial derivatives. The presence of two antiparallel sperm bundles per cyst also in Meloidae and Rhipiphoridae suggests that it is a strong trait synapomorphic for Tenebrionoidea. The huge degeneration of whole sperm cells in several cysts of testes during spermiogenesis is also described.

  17. Penicillamine prevents ram sperm agglutination in media that support capacitation.

    PubMed

    Leahy, T; Rickard, J P; Aitken, R J; de Graaf, S P

    2016-02-01

    Ram spermatozoa are difficult to capacitate in vitro. Here we describe a further complication, the unreported phenomenon of head-to-head agglutination of ram spermatozoa following dilution in the capacitation medium Tyrodes plus albumin, lactate and pyruvate (TALP). Sperm agglutination is immediate, specific and persistent and is not associated with a loss of motility. Agglutination impedes in vitro sperm handling and analysis. So the objectives of this study were to investigate the cause of sperm agglutination and potential agents which may reduce agglutination. The percentage of non-agglutinated, motile spermatozoa increased when bicarbonate was omitted from complete TALP suggesting that bicarbonate ions stimulate the agglutination process. d-penicillamine (PEN), a nucleophilic thiol, was highly effective at reducing agglutination. The inclusion of 250 μM PEN in TALP reduced the incidence of motile, agglutinated spermatozoa from 76.7 ± 2.7% to 2.8 ± 1.4%. It was then assessed if PEN (1 mM) could be included in existing ram sperm capacitation protocols (TALP +1 mM dibutyryl cAMP, caffeine and theophylline) to produce spermatozoa that were simultaneously capacitated and non-agglutinated. This protocol resulted in a sperm population which displayed high levels of tyrosine phosphorylated proteins and lipid disordered membranes (merocyanine-540) while remaining motile, viable, acrosome-intact and non-agglutinated. In summary, PEN (1 mM) can be included in ram sperm capacitation protocols to reduce sperm agglutination and allow for the in vitro assessment of ram sperm capacitation.

  18. [Sperm-antibodies - practical importance in the male fertility disorder].

    PubMed

    Meili, H U; Bandhauer, K

    1976-07-01

    On the basis of abundant statistics it is known that in about 5% of infertile males fertility-inhibiting antibodies are present, which can lead to immobilization or agglutination of the sperm; they can block acrosome activity or become cytotoxically active. The motility of spermatozoon charged with antibodies and partially immobilized or agglutinized is probably not sufficient to penetrate the cervical mucus to reach the egg. Immunological sterility can be suspected in certain situations: infertility in a couple where there is no apparent cause of female infertility; anamnestic or clinical indication of chronic prostatitis, vesiculitis, or epididymitis; spontaneous agglutination or motility reduction in the spermiogram (not demonstrable in all cases); pathological postcoital test by the Sims-Huhner method. Since the last is only 50% reliable, diagnosis of antibodies is dependent on laboratory tests such as: micro-sperm-agglutination test, macro-sperm-agglutination test, sperm immobilization test, hema-agglutination test, and capillary X-ray. As yet there is no satisfactory treatment for this type of male fertility disorder. The only promising results in this area are achieved when the inflamed source for the antigen-antibody reaction is found and removed. Diagnosis of sperm antibodies in male infertility, however, can be the clear indication for heterologic insemination.

  19. Novel gamete receptors that facilitate sperm adhesion to the egg coat.

    PubMed

    Ensslin, Michael A; Lyng, Robert; Raymond, Adam; Copland, Susannah; Shur, Barry D

    2007-01-01

    Mammalian fertilization is initiated by species-specific binding of the sperm to the zona pellucida, or egg coat. Previous studies suggested that sperm adhesion to the egg coat is facilitated, at least in part, through the binding of sperm surface beta1 ,4-galactosyltransferase I (GaIT) to glycoside chains on the egg coat glycoprotein, ZP3. Binding of multiple ZP3 oligosaccharides induces aggregation of GaIT within the sperm membrane, triggering, directly or indirectly, a pertussis toxin sensitive G-protein cascade leading to induction of the acrosome reaction. Consistent with this, spermatozoa bearing targeted deletions in GaIT are unable to bind ZP3 or undergo ZP3-dependent acrosomal exocytosis; however, unexpectedly, GaIT-null sperm are still able to bind to the egg coat. This indicates that sperm-egg binding requires at least two independent binding mechanisms; a GaIT-ZP3-independent event that mediates initial adhesion, followed by a GaIT-ZP3 interaction that facilitates acrosomal exocytosis. Our recent efforts have focused on the identification and characterization of these novel gamete receptors. One recently identified sperm protein that is required for sperm adhesion to the egg coat is SED1. SED1 is a bimotif protein composed of two Notch-like EGF repeats and two discoidin/complement F5/8 domains. SED1 is secreted by the epididymal epithelium and coats spermatozoa as they progress through the epididymis. Spermatozoa null for SED1 fail to bind the egg coat, illustrating its requirement for gamete adhesion. Interestingly, SED1 is also expressed by a variety of other epithelial tissues, where it appears to be required for epithelial morphogenesis and/or maintenance. A second novel gamete receptor has recently been identified on the coat of ovulated oocytes. This ZP3-independent, egg coat component is a high molecular weight, wheat germ agglutinin (WGA)-reactive glycoprotein that is derived from oviduct secretions and appears to participate in initial sperm

  20. Computer-assisted sperm analysis of fresh epididymal cat spermatozoa and the impact of cool storage (4 degrees C) on sperm quality.

    PubMed

    Filliers, M; Rijsselaere, T; Bossaert, P; De Causmaecker, V; Dewulf, J; Pope, C E; Van Soom, A

    2008-12-01

    Epididymal cat sperm is commonly used for in vitro fertilization. Because of the high variability in preparation protocols and methods of evaluation, sperm quality may vary considerably between experiments and laboratories. The aims of the present study were (1) to describe an epididymal sperm preparation protocol to produce clean, highly motile samples using density gradient centrifugation, (2) to provide reference values of computer-assisted semen analysis (CASA) parameters of fresh epididymal cat sperm after density gradient centrifugation and (3) to investigate the effect of cool storage on various spermatozoa characteristics. After slicing the epididymides, viable and motile sperm cells were isolated using Percoll centrifugation. Sperm motility parameters were subsequently assessed using CASA in experiment 1. In experiment 2, fresh (day 0) sperm samples were evaluated for motility parameters (HTR) and stained for assessment of acrosomal status (FITC-PSA), morphology (eosin/nigrosin (E/N)), membrane integrity (E/N and SYBR((R))14-PI) and DNA fragmentation (TUNEL). After addition of a Tris-glucose-citrate diluent containing 20% egg yolk, samples were cooled to 4 degrees C and reassessed on d1, d3, d5, d7 and d10. Cool storage impaired most motility and velocity parameters: MOT, PMOT, VAP, VSL, VCL, BCF, RAPID and the percentage of normal spermatozoa showed a decrease over time (P<0.05) as compared to fresh samples. In contrast, STR, ALH, membrane integrity, DNA fragmentation and the percentage of acrosome intact spermatozoa were not affected by cool storage. However, the influence of cool storage of cat spermatozoa on subsequent in vitro embryo development and quality after IVF requires further investigation.

  1. Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders.

    PubMed

    Lange-Consiglio, A; Meucci, A; Cremonesi, F

    2013-01-01

    The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r(2)>0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P<0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage.

  2. Fluorescent multiple staining and CASA system to assess boar sperm viability and membranes integrity in short and long-term extenders

    PubMed Central

    Lange-Consiglio, A.; Meucci, A.; Cremonesi, F.

    2013-01-01

    The aim of this study was to assess the effect on boar spermatozoa quality of in vitro storage in short and long-term extenders by fluorescent multiple staining (FMS) and computer assisted semen analyzer (CASA). Fresh ejaculates from three healthy, sexually mature boars were diluted with equal volumes of six short-term or three long-term commercial extenders and stored at 19°C for 6 days (short-term) or 12 days (long-term). The integrity of spermatozoa membranes was analyzed by FMS using propidium iodide, 5,5’,6,6’-tetrachloro-1,1’,3,3’ tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) and fluorescein isothiocyanate-conjugated peanut agglutinin (PNA). The results obtained from this staining were compared with spermatozoa motility assessed by CASA. Our study showed that the number of viable spermatozoa with non-reacted acrosomes and intact mitochondria was positively correlated with the rate of motile spermatozoa (r2>0.9) irrespective of the extender used. In all extenders the number of motile spermatozoa significantly decreased as preservation period increased (P<0.05). FMS test is a potent indicator of sperm motility because it analyses mitochondrial integrity independently from observable alterations in motility. The best performing extenders were BTS for short-term storage and TRI-x-Cell for long-term storage. PMID:26623308

  3. Changes in sperm quality and lipid composition during cryopreservation of boar semen.

    PubMed

    Maldjian, A; Pizzi, F; Gliozzi, T; Cerolini, S; Penny, P; Noble, R

    2005-01-15

    Egg yolks are commonly used in diluents in order to improve the freezability of semen. Two aspects of the role of lipids in boar semen freezability are reported in this article. The first one concerns the eventual exchanges of lipid components between the spermatozoa and the yolk-based diluent during cryopreservation. Two types of yolk have been considered as ingredients in diluents for cryopreservation: yolks with a standard fatty acid composition and yolks enriched in docosahexaenoic acid (DHA). The relation between lipid exchanges and the quality of fresh semen is considered. The other aspect concerns the possibility to enhance the freezability of boar spermatozoa by altering the plasma membranes under the influence of dietary fatty acids. Sperm