Showing papers on "Sperm plasma membrane published in 2000"

The capacitating agent bicarbonate induces protein kinase A-dependent changes in phospholipid transbilayer behavior in the sperm plasma membrane.

Abstract: A flow cytometric procedure was used to follow the effect of bicarbonate, a key inducer of sperm capacitation in vitro, on the transbilayer behavior of C6NBD-phospholipids in the plasma membrane of living acrosome-intact boar spermatozoa under physiological conditions. In the absence of bicarbonate, 97% of C6NBD-phosphatidylserine and 78% of C6NBD-phosphatidylethanolamine was rapidly translocated from the outer leaflet to the inner, whereas relatively little C6NBD-phosphatidylcholine and C6NBD-sphingomyelin was translocated (15% and 5%, respectively). Inclusion of 15 mM bicarbonate/5%CO(2) markedly slowed down the rates of translocation of the aminophospholipids without altering their final distribution, whereas it increased the proportions of C6NBD-phosphatidylcholine and C6NBD-sphingomyelin translocated (30% and 20%, respectively). Bicarbonate activated very markedly the outward translocation of all four phospholipid classes. The changes in C6NBD-phospholipid behavior were accompanied by increased membrane lipid disorder as detected by merocyanine 540, and also by increased potential for phospholipase catabolism of the C6NBD-phospholipid probes. All three changes were mediated via a cAMP-dependent protein phosphorylation pathway. We suspect that the changes result from an activation of the non- specific bidirectional translocase ('scramblase'). They have important implications with respect to sperm fertilizing function.

Seminal plasma proteins revert the cold-shock damage on ram sperm membrane.

Abstract: Ejaculated ram spermatozoa, freed from seminal plasma by a dextran/swim-up procedure and exposed to cold shock, were incubated with ram seminal plasma proteins and analyzed by fluorescence markers and scanning electron microscopy. Seminal plasma proteins bound to the sperm plasma membrane modified the functional characteristics of damaged spermatozoa, reproducing those of live cells. Scanning electron microscopy showed that the dramatic structural damage induced by cooling reverted after incubation with seminal plasma proteins. Assessment of membrane integrity by fluorescence markers also indicated a restoration of intact-membrane cells. This protein adsorption is a concentration-dependent process that induces cell surface restoration in relation to the amount of protein in the incubation medium. Fractionation of ram seminal plasma proteins by exclusion chromatography provided three fractions able to reverse the cold shock effect. Scanning electron microscopy also confirmed the high activity of one fraction, because approximately 50% of cold-shocked sperm plasma membrane surface was restored to its original appearance after incubation. Differences in composition between the three separated fractions mainly resulted from one major band of approximately 20 kDa, which must be responsible for recovering the sperm membrane permeability characteristic of a live cell.

Intracellular events and signaling pathways involved in sperm acquisition of fertilizing capacity and acrosome reaction.

Abstract: Two processes, namely capacitation and acrosome reaction, are of fundamental importance in the fertilization of oocyte by spermatozoon. Physiologically occurring in the female genital tract, capacitation is a complex process, which renders the sperm cell capable for specific interaction with the oocyte. During capacitation, modification of membrane characteristics, enzyme activity and motility properties of spermatozoa render these cells able to penetrate oocyte investments and responsive to stimuli that induce acrosome reaction prior to fertilization. Physiological acrosome reaction occurs upon interaction of the spermatozoon with the zona pellucida protein ZP3. This is followed by liberation of several acrosomal enzymes and other constituents that facilitate penetration of the zona and expose molecules on the sperm equatorial segment that allows fusion of sperm membrane with the oolemma. The molecular mechanisms and the signal transduction pathways mediating the processes of capacitation and acrosome reaction have been partially defined, and appear to involve modifications of intracellular calcium and other ions, lipid transfer and phospholipid remodeling in sperm plasma membrane as well as changes in protein phosphorylation. Some of the kinases and phosphorylated proteins that are involved in the processes of capacitation and acrosome reaction have been now characterized. Characterization of sperm receptors to physiological inducers of acrosome reaction is in progress. This review summarizes the main signal transduction pathways involved in capacitation and acrosome reaction.Furthermore, the mechanisms underlying sperm DNA fragmentation are also briefly reviewed.

The effect of extracellular ice and cryoprotective agents on the water permeability parameters of human sperm plasma membrane during freezing

Abstract: A firm biophysical basis for the cryopreservation of human spermatozoa is limited by a lack of knowledge regarding the water permeability characteristics during freezing in the presence of extracellular ice and cryoprotective agents (CPA). Cryomicroscopy cannot be used to measure dehydration during freezing in human spermatozoa because of their highly non-spherical shape and their small dimensions which are at the limits of light microscopic resolution. Using a new shape-independent differential scanning calorimeter (DSC) technique, volumetric shrinkage during freezing of human sperm cell suspensions was obtained at cooling rates of 5 and 10 degrees C/min in the presence of extracellular ice and CPA. Using previously published data, the human sperm cell was modelled as a cylinder of length 40.2 micrometer and a radius of 0.42 micrometer with an osmotically inactive cell volume, V(b), of 0.23V(o), where V(o) is the isotonic cell volume. By fitting a model of water transport to the experimentally obtained volumetric shrinkage data, the best fit membrane permeability parameters (L(pg) and E(Lp)) were determined. The 'combined best fit' membrane permeability parameters at 5 and 10 degrees C/min for human sperm cells in modified media are: L(pg) = 2. 4x10(-14) m(3)/Ns (0.14 micrometer/min-atm) and E(Lp) = 357.7 kJ/mol (85. 5 kcal/mol) (R(2) = 0.98), and in CPA media (with 6% glycerol and 10% egg yolk) are L(pg)[cpa] = 0.67x10(-14) m(3)/Ns (0.04 micrometer/min-atm) and E(Lp)[cpa] = 138.9 kJ/mol (33.2 kcal/mol) (R(2) = 0.98). These parameters are significantly different from previously published parameters for human spermatozoa obtained at suprazero temperatures and at subzero temperatures in the absence of extracellular ice. The parameters obtained in this study also suggest that damaging intracellular ice formation (IIF) could occur in human sperm cells at cooling rates as low as 25-45 degrees C/min, depending on the concentrations of the CPA. This may help to explain the discrepancy between the empirically determined optimal cryopreservation cooling rates ( 7000 degrees C/min) obtained using previously published suprazero human sperm permeability parameters which do not account for the presence of extracellular ice.

Sperm Calcium Levels and Chlortetracycline Fluorescence Patterns are Related to the In Vivo Fertility of Cryopreserved Bovine Semen

Abstract: Cryopreserved bovine semen is less fertile than fresh semen for reasons that have not been fully elucidated. Cryopreservation is known to disrupt the sperm plasma membrane and it induces premature capacitation of a sperm subpopulation, which may be a result of the increased internal calcium levels after thawing. To test the hypothesis that sperm intracellular calcium level is correlated with in vivo fertility, we used the fluorescent calcium indicator, indo-1, and flow cytometry to assess intracellular calcium levels in frozen-thawed sperm from bulls of varying degrees of fertility. We also tested a second hypothesis that the physiological status of sperm, as assessed by the chlortetracycline (CTC) fluorescent assay, is correlated with fertility. As detected by indo-1 fluorescence, the intracellular calcium level is negatively correlated with bull fertility immediately after thawing (P = .0362; n = 3 ejaculates from each of 10 animals). Moreover, there was a significant difference between the 3 most and least fertile bulls over 4 hours of incubation (P < .05; n = 3 ejaculates per bull). Finally, there was a positive correlation between sperm displaying the CTC acrosome reaction pattern and fertility (P = .0014; n = 3 ejaculates from each of 10 bulls).

Effect of cholesterol on the motility and plasma membrane integrity of frozen equine spermatozoa after thawing.

Abstract: The aim of the present study was to investigate the cryoprotectant properties of cholesterol after incorporation into the plasma membranes of equine spermatozoa. A cholesterol-methyl-beta-cyclodextrin complex was used to alter sperm plasma membrane cholesterol content. Ejaculates from six stallions were centrifuged in a non-fat skimmed milk glucose-sucrose extender (MK) or a modified Tyrode's medium (TALP). The sperm pellets were resuspended in the appropriate extender with or without added cholesterol (0.125 mmol cholesterol-methyl-beta-cyclodextrin complex l(-1)) and incubated at 24 degrees C for 15 min. After incubation, the aliquots were centrifuged and the sperm pellets were resuspended in lactose-EDTA-egg yolk freezing extender, frozen in static nitrogen vapour and stored at -196 degrees C. The straws were thawed and the motility and plasma membrane integrity of the spermatozoa were analysed. Addition of cholesterol to the incubation extenders improved the mean percentages of motile, progressively motile and rapidly motile spermatozoa in both the MK and TALP extenders containing cholesterol compared with extenders without cholesterol (P < 0.05). The percentage of spermatozoa with intact plasma membranes was higher in samples incubated in extenders containing cholesterol than in those without cholesterol (P < 0.05). The results of this study indicate that cyclodextrins can be used to incorporate cholesterol into equine sperm plasma membranes and that cholesterol incorporation imparts protection to the spermatozoa during cryopreservation.

Source of dietary lipid affects sperm plasma membrane integrity and fertility in rainbow trout Oncorhynchus mykiss (Walbaum) after cryopreservation

Abstract: The effects of dietary lipid from four experimental diets on the fatty acid (FA) composition and cholesterol (CHOL) content of spermatozoa and spermatozoal plasma membranes and their consequences for sperm viability after cryopreservation were evaluated in rainbow trout Oncorhynchus mykiss (Walbaum). The four sources of lipid were herring oil (adequate n-3 FA), menhaden oil (high n-3 FA), safflower oil (high n-6 FA) or tallow (high saturated FA), and they comprised 12% of the total diet. Spermatozoa from fish fed the tallow diet had significantly (P < 0.05) higher CHOL levels than spermatozoa from the fish fed the other diets. The spermatozoal plasma membranes from fish fed the tallow diet had significantly (P < 0.05) higher CHOL and monounsaturated fatty acid levels than those from fish fed the menhaden or safflower oil diets, but were not different from membranes of fish fed the herring oil diet. Cryopreserved spermatozoa from fish fed the tallow or herring oil diets exhibited less membrane damage (P < 0.05) and produced a higher percentage (P < 0.05) of eyed embryos compared with spermatozoa from the menhaden or safflower oil-fed fish. Therefore, it would appear that high levels of CHOL and monounsaturated fatty acids provided the spermatozoa with increased resistance to cryopreservation damage.

Antioxidative effect of melatonin on human spermatozoa.

Abstract: The ability of melatonin to suppress experimentally induced lipid peroxidation (LPO) in sperm membrane was investigated in 41 samples of infertile men. Iron/ascorbate (0.04/0.2 mmol)-induced LPO was measured by the formation of malondialdehyde (MDA) using the thiobarbituric acid method. Sperm incubated in the presence of melatonin (2-6 mmol) exhibited a concentration-dependent decrease of MDA generated from hydroperoxide of the sperm plasma membrane in the presence of promoter system. Addition of 6 mmol of melatonin significantly reduced the rate of lipid peroxidation in sperm of unselected donors (mean +/- SE in control samples = 26.4 +/- 2.9 vs. 6.5 +/- 1.1 nmol MDA/108 sperm in melatonin-treated samples; n = 16, p <. 005). Inhibitory effect of melatonin was also significant in the presence of 0.015 mmol of ferrous ions (20.5 +/- 1.7 vs. 7.9 +/- 1.6 nmol MDA/108 sperm in melatonintreated samples; n = 7, p <. 02) and 0.005 mmol of ferrous ions (20.2 +/- 2.8 vs. 9.9 +/- 2.4 nmol MDA/ 108 sperm; n = ...

Cellular and molecular biology of capacitation and acrosome reaction in spermatozoa.

Abstract: A comparative account is given of advances in cellular and molecular biology of capacitation and acrosome reaction in spermatozoa by comparing and contrasting their biochemical and physiological changes in response to various factors in vivo and in vitro. It can now be stated that phenomena of sperm capacitation and acrosome reaction are endogenous molecular events occurring at the membrane level which can be modulated by external environmental factors. The molecular mechanisms and the signal transduction pathways mediating the process of capacitation and acrosome reaction are only partially defined and appear to involve modification of intracellular Ca2+ and other ions, lipid transfer, and phospholipid remodeling in the sperm plasma membrane as well as changes in protein phosphorylation. Evidences for the involvement of cAMP-dependent kinase pathway in the acrosome reaction are discussed. The mediation of one or more external signals by the sperm plasma membrane appears to activate this pathway after or simultaneously with the influx of Ca2+. Concurrent with or following entry of Ca2+, adenylate cyclase is activated, leading to increased concentrations of cAMP-activation of cAMP-dependent kinase and protein phosphorylation; the identity of such proteins and their role in the acrosome reaction must be determined. The roles of biological effectors of the acrosome reaction, such as ZP3 and follicular fluid are still to be defined at the molecular level. The gaps in our knowledge about the cellular and molecular aspects of capacitation and acrosome reaction are emphasized.

Measurement of intracellular calcium concentration and plasma membrane potential in human spermatozoa using flow cytometry.

Abstract: We report 2 novel approaches using flow cytometry to measure intracellular calcium concentration and plasma membrane potential in human spermatozoa. Both approaches have the potential to measure different responses in subpopulations of cells, which is particularly useful when studying heterogeneous populations such as human spermatozoa. Intracellular calcium concentration ([Ca2+]i) was measured using the probe indo-1/AM. This allowed measurements to be made that were independent of variation in cell size and dye loading. It also enabled dead cells to be directly identified and excluded from the analyses without the need for counterstaining. Mean basal [Ca2+]i was determined as 50 nM (25-75 nM range) and, in response to the agonist progesterone (20 microM), this increased transiently to 195 nM (125-285 nM range) before declining to approximately half the maximal level within 2 minutes (values in parentheses correspond to the range of values typically found within a sperm population from 1 sample). These results are comparable with previously published data on whole sperm populations. Sperm membrane potential (VM) was assayed using the probe DiOC6(3). In carefully controlled experiments, a marked depolarization of the plasma membrane potential of capacitated spermatozoa was observed in response to progesterone (20 microM). Following in vitro capacitation, the sperm plasma membrane potential became hyperpolarized compared with the noncapacitated state. Therefore, this technique may be used to assay for sperm capacitation in vitro.

Potential role of alphav and beta1 integrins as oocyte adhesion molecules during fertilization in pigs.

Abstract: Integrin molecules are cell adhesion molecules that are thought to be involved in sperm‐oocyte interaction in rodents and humans. The objective of this study was to evaluate whether integrin molecules were present on the surface of pig oocytes, consistent with involvement in sperm‐oocyte interaction in this species. Immunocytochemistry and confocal microscopy were used to evaluate the presence of β1, and α1, α2, α3, α4, α5, α6 and αv integrin subunits on the plasma membrane of pig oocytes. The β1 and αv integrin subunits were present consistently at the surface of pig oocytes; however, the remaining α integrin subunits evaluated were not routinely detected. The antibodies to the β1 and αv integrin subunits recognized appropriately sized protein bands on western blots of partially purified oocyte plasma membrane. These two antibodies also recognized oocyte plasma membrane protein isolated from a sperm plasma membrane affinity column. Sperm plasma membrane proteins of 137 and 93 kDa appeared to be the ligands for the β1 integrin subunit as revealed by a western sandwich blot. Antibody to an extracellular domain of the β1 integrin subunit reduced pig sperm‐oocyte binding (P < 0.05), also indicating an assisting role for a β1 oocyte integrin subunit in sperm‐oocyte interaction in pigs. These results are consistent with an αvβ1 pig oocyte integrin interacting with a ligand on the sperm plasma membrane during fertilization.

Cholesterol Inhibitory Effects on Human Sperm—Induced Acrosome Reaction

Abstract: Progesterone (P 4 ) is known to induce an acrosome reaction in mammalian sperm in vitro, whereas cholesterol is a major inhibitor of acrosome reaction. This study had three objectives: to study the in vitro effects of exogenous cholesterol on acrosome reactions in human sperm, to study the mechanism by which cholesterol affects P 4 -induced acrosome reaction and those induced by dibutyryl cyclic adenosine monophosphate (db-cAMP), and to study the status of the P 4 surface receptor during capacitation and acrosome reaction and its relationship with cholesterol and different acrosome reaction inducers. Acrosome reaction was induced with exposure to 10 μg/mL of P 4 for 30 minutes and 1 mM of db-cAMP for 30 minutes in motile sperm either in the presence or absence of 0.1-1 μg/mL of cholesterol for 30 minutes. The effects of a 30-minute exposure to 1 μg/mL of β-sitosterol, a cholesterol plant analogue, as well as the effects of cholesterol on P 4 -induced acrosome reactions were compared. Fluorescein isothiocyanate-labeled albumin-progesterone conjugate (P 4 -FITC-BSA) was used as the probe in order to quantify the percentage of sperm in which the P 4 surface receptor was exposed. The results of this study indicate that cholesterol inhibited P 4 -induced acrosome reactions when added to the sperm during capacitation (long incubation) and when it was added with P 4 during the induction of acrosome reactions (short incubation). Similarly, acrosome reaction that was induced by db-cAMP was also inhibited by cholesterol. Fifty percent of P 4 -induced acrosome reaction was inhibited by a cholesterol concentration of 0.2 μg/mL. Cholesterol's inhibition of induced acrosome reaction was independent of P 4 concentration. β-Sitosterol inhibited P 4 -induced acrosome reaction in a dose-dependent manner that was identical to that of cholesterol. We observed that increases in the P 4 surface receptor exposure were time-dependent and receptors migrated toward the equatorial segment during the first 2 hours of capacitation. We also found that db-cAMP induced the appearance of the P 4 surface receptor in the sperm plasma membrane and that cholesterol inhibited it. The results of this study suggest that cholesterol inhibits acrosome reaction in a noncompetitive manner by modifying the structure of the sperm plasma membrane, which prevents exposure of the P 4 surface receptor for P 4 binding.

Sperm-egg interaction at fertilization: glycans as recognition signals.

Abstract: This article first examines the events occurring in male and female genital tracts, which prepare human sperm to encounter the egg. Central is a glycoprotein, gp20, homologous to the leukocyte antigen CD52. This protein is secreted in the epididymal cells, inserted in the sperm plasma membrane and exposed in the equatorial region of the head at the end of the capacitation process. The mechanisms and molecules of the first interaction event between gametes in the mollusk bivalve Unio elongatulus and the current state of our knowledge of the same interaction in other species is then considered. The egg of Unio is very peculiar because it is highly polarized. Similar to other well-known egg models, the ligand for recognition is located on the egg coat which is a sort of fibrous network made up of very few glycoproteins, while the receptor is on the sperm surface. The difference is that in this egg, the ligand molecules are not uniformly distributed but are restricted to an area of the egg coat at the vegetal pole, the crater area. The role of carbohydrates in ligand function and of a specific type of oligosaccharide chain in particular, is discussed in the wider context of glycans acting as recognition signals.

Biochemical Characterization of Two Ram Cauda Epididymal Maturation-Dependent Sperm Glycoproteins

Abstract: Rabbit polyclonal antibodies were raised against ram cauda epididymal sperm proteins solubilized by N-octyl-b-D-glucopyranoside (anti-CESP) and against proteins of the fluid obtained from the cauda epididymidis (anti-CEF). The anti-CESP polyclonal antibody reacted with several bands from 17 to 111 kDa with different regionalization throughout the epididymis. The strongest epitopes at 17 kDa and 23 kDa were restricted to the cauda epididymidis. The anti-CEF polyclonal antibody reacted mainly with a 17-kDa and a 23-kDa compound in the cauda sperm extract. These cauda epididymal 17- and 23-kDa proteins disappeared after orchidectomy, but they reappeared in the same regions after testosterone supplementation, indicating that they were secreted by the epithelium. The fluid and membrane 17- and 23-kDa antigens had a low isoelectric point and were glycosylated. The fluid 17- and 23kDa proteins had hydrophobic properties: they were highly enriched in the Triton X-114 detergent phase and could be extracted from the cauda epididymal fluid by a chloroform-methanol mixture. These proteins were further purified, and their N-terminal sequences did not match any protein in current databases. A polyclonal antibody against the fluid 17-kDa protein recognized the protein in the cauda epididymal sperm extract and immunolocalized it on the sperm flagellum membrane and at the luminal border of all cells in the cauda epididymal epithelium. These results indicated that secreted glycoproteins with hydrophobic properties could be directly integrated in a specific domain of the sperm plasma membrane.

Maturation-dependent glycoproteins containing both N- and O-linked oligosaccharides in epididymal sperm plasma membrane of rhesus monkeys (Macaca mulatta).

Abstract: Glycosylation is one of the important post-translational modifications of sperm plasma membrane proteins during the maturation of epididymal spermatozoa that results in the development of motility and fertilizing capability. The aim of the present study was to identify and characterize the maturation-dependent asparagine-linked (N-linked) and serine- and threonine-linked (O-linked) glycoproteins of the epididymal spermatozoa of rhesus monkeys. The presence of N- and O-linked glycoproteins was confirmed by treatment of sperm membranes with N-glycosidase F and O-glycosidase. The major maturation-dependent sperm membrane glycoproteins identified on blots of SDS-PAGE-fractionated proteins of purified sperm plasma membranes from five segments of epididymis, probed with biotinylated lectins and Vectastain-ABC reagent included O-linked 170, 150, 86 and 60/58 kDa glycoproteins; N-linked 68, 56, 48 and 38 kDa glycoproteins and N- and O-linked 116 kDa glycoprotein, all of which exhibited marked differences in the degree of glycosylation between immature and mature sperm surfaces. These glycoproteins can be used as markers of sperm maturation in the epididymis of rhesus monkeys, during the screening of antifertility agents acting at the epididymis, or may be developed as potential sperm antigens. The 100% inhibition of fertility in female rats and rabbits immunized with major maturation-dependent 116 kDa glycoprotein showed the significance of glycosylation changes in the maturation status of epididymal spermatozoa. This 116 kDa protein can be used as a marker parameter of sperm maturation in the rhesus monkey, which is often the preferred animal model for preclinical studies. These results will contribute to the identification of an appropriate animal model for the development of male contraceptives in humans.

Metal ions and human sperm mannose receptors.

Abstract: Zinc and lead concentrations were measured in seminal plasma from fertile donors, infertile men with varicocoele and men undergoing work-ups for in vitro fertilization. Ejaculated spermatozoa from these subjects were incubated in vitro with various metal ions and/or dibromoethane and dibromochloropropane. Mannose receptor expression was correlated with metal and toxicant levels. Sperm distributions of potassium channels were compared with lead ions and calcium channels with zinc ions. Mannose receptor expression by capacitated spermatozoa increased linearly with seminal plasma zinc levels, and correlated inversely with lead levels. Cobalt had no effect on mannose receptor expression, but nickel had a concentration-dependent biphasic effect. Mannose receptor expression was not affected by dibromoethane and dibromochloropropane if the cholesterol content of the sperm membrane was high, but mannose receptor expression was decreased in low cholesterol spermatozoa by exposures below estimated permissive exposure limits. Potassium channels and lead ions co-localized over the entire head of human spermatozoa, while both calcium channels and zinc ions were confined to the equatorial segment of the head. Mannose receptor expression on the external surface of the human sperm plasma membrane is a biomarker for the effects of transition and heavy metals and organic toxicants on sperm fertility potential.

A Universal Bicarbonate Sensor

Abstract: The life-span of sperm may be short but it is certainly busy. The three principal molecular events that prepare sperm for fertilization are all controlled by the intracellular nucleotide adenosine 3',5'-monophosphate (cAMP). One of these, capacitation, is also regulated by bicarbonate ions. The elusive connection between cAMP and bicarbonate ions now appears to be solved as [Kaupp and Weyand][1] explain in their Perspective. Bicarbonate ions enter sperm through the anion transporter in the sperm plasma membrane and activate the soluble form of adenylyl cyclase, the enzyme that synthesizes cAMP ([ Chen et al .][2]) [1]: http://www.sciencemag.org/cgi/content/full/289/5479/559 [2]: http://www.sciencemag.org/cgi/content/short/289/5479/625

Assessing equine sperm-membrane integrity.

Abstract: Summary. The swelling of cells in a hypoosmotic medium has been described as an important criterion for assessing the functional integrity of the sperm plasma membrane. The resistance of equine spermatozoa to osmolarity changes was studied by extending 98 semen samples collected from nine stallions in media at five osmolarities (300, 200, 150, 100, and 50 mOsmol l−1). The response of the cells was measured by the spermatocrit technique and eosin staining. Spermatocrit determines the increase on spermatozoal volume under hypo-osmotic conditions, a sign of functional integrity of sperm plasma membrane, whereas the eosin staining evaluates the viability of spermatozoa. A significant positive correlation (P<0.01) was observed between spermatocrit values and percentage of eosin-unstained cells. Spermatocrit measurements and eosin staining proved to be useful methods to evaluate the integrity of sperm plasma membrane under hypoosmotic conditions and could be used as an additional criterion to predict semen preservation ability.

Effects of cryopreservation on progesterone-induced ion fluxes and acrosome reaction in human spermatozoa

Abstract: The present study evaluated the effects of cryopreservation on progesterone-induced variations of calcium ion concentration [Ca 2+ ] i , plasma membrane potential and acrosome reaction in human spermatozoa. Spermatozoa from 10 fertile donors were divided in two equivalent aliquots, one used as control (fresh spermatozoa) and the other used after freezing-thawing. Measurement of spermatozoa [Ca 2+ ] i before and after freezing-thawing showed a significant reduction of basal [Ca 2+ ] i in thawed spermatozoa (P < 0.01). Progesterone induced a rise of [Ca 2+ ] i both in fresh and thawed spermatozoa with a significant reduction after freezing-thawing (P < 0.01). The monitoring of sperm plasma membrane potential demonstrated that progesterone induced plasma membrane depolarization in fresh spermatozoa that was absent in thawed spermatozoa. The inhibitory effects of freezing-thawing on progesterone induced [Ca 2+ ] i and plasma membrane potential variations in human spermatozoa were closely related to the inhibition of the acrosome reaction. In conclusion the present study demonstrates that freezing-thawing procedures reduce the responsiveness of human spermatozoa to progesterone in terms of [Ca 2+ ] i rise and completely inhibit its effects on plasma membrane potential variations, thus supporting the hypothesis that freezing-thawing procedures may differently modify the plasma membrane receptors for progesterone in human spermatozoa which are known to express at least two receptors for this steroid in their plasma membrane.

Changes in the plasma membrane proteins of stallion spermatozoa during maturation in the epididymis.

Abstract: The present paper reports modifications in the electrophoretic and cytochemical characteristics of mature and immature stallion spermatozoa. Some sperm surface glycoproteins (36, 32, 29, 21, 20, 18 kDa) detected in cauda epididymidis spermatozoa, were either absent or present in a very low relative concentration in immature sperm cells. A major 14 kDa protein band, observed in sperm extracts obtained from ductus efferentes, progressively decreased along the epididymal ductus. The nature and distribution of carbohydrate residues on the sperm membrane, during epididymal maturation, was also studied by use of lectin probes. Some protein bands bound concanavalin A while others, as the 36, 32 and 20 kDa proteins, exhibited higher affinity for WGA lectin. The distribution and relative density of mannose-, galactose-, N-acetylglucosamine-, N-acetylgalactosamine-, fucose- and sialic acid-containing macromolecules showed a characteristic pattern depending on the sperm membrane domain and on its origin. Some sperm surface domains displayed affinity for more than one lectin, indicating a diversity in their exposed carbohydrate residues, whereas others bound only one or no lectin. The passage of spermatozoa through the epididymidis was accompanied by changes in the accessibility or abundance of lectin ligands. Some lectins (UEA, WGA, LPA) gave stronger reaction in mature spermatozoa, while others (RCA, WFH, PNA) stained better immature spermatozoa. This remodeling of sperm surface molecules is probably a consequence of interactions between spermatozoa and the epididymal secretions, and may reflect addition or adsorption of new molecules, space configurations changes or biochemical modifications of pre-existing compounds. Our results suggest that the distribution and density of terminal oligosaccharidic residues on the sperm plasma membrane have species-specific characteristics. These post testicular developmental changes may be of significance in the overall understanding of the stallion fertility.

Carbohydrates mediate sperm-ovum adhesion and triggering of the acrosome reaction.

Abstract: The fertilization process is the net result of a complex sequence of events that collectively result in the fusion of the opposite gametes. The male gamete undergoes continuous morphological and biochemical modifications during sperm development in the testis (spermatogenesis), maturation in the epididymis, and capacitation in the female reproductive tract. Only the capacitated spermatozoa are able to recognize and bind to the bioactive glycan residue(s) on the ovum's extracellular coat, the zona pellucida (ZP). Sperm-zona binding in the mouse and several other species is believed to take place in two stages. First, capacitated (acrosome-intact) spermatozoa loosely and reversibly adhere to the zona-intact ovum. In the second stage tight irreversible binding occurs. Both types of bindings are attributed to the presence of glycan - binding proteins ( receptors ) on the sperm plasma membrane and their complementary bioactive glycan units ( ligands) on the surface of the ZP. The carbohydrate-mediated adhesion event initiates a signal transduction cascade resulting in the exocytosis of acrosomal contents. This step is believed to be prerequisite which allows the hyperactivated acrosome-reacted spermatozoa to penetrate the ZP and fertilize the ovum. This review focuses on the role of carbohydrate residues in sperm-ovum interaction, and triggering of the acrosome reaction. I have attempted to discuss extensive progress that has been made to enhance our understanding of the well programmed multiple molecular events necessary for successful fertilization. This review will identify these events, and discuss the functional significance of carbohydrates in these events.

Ultrastructure of sperm development in the free-living marine nematodes of the family Chromadoridae (Chromadorida: Chromadorina).

Abstract: Spermatogenesis in two species of free-living marine nematodes from the family Chromadoridae (Panduripharynx pacifica and Euchromadora robusta) was studied electron-microscopically The spermatogonia of both species are undifferentiated polygonal cells with a large nucleus surrounded by a small amount of cytoplasm In P pacifica the cytoplasm of spermatocytes contains many Golgi bodies, cisternae of RER, ribosomes, mitochondria and dense spherical bodies Filamentous material is accumulated in spermatids, which contain only mitochondria and a fragmented (or lobed) nucleus devoid of the nuclear envelope The immature sperm resembles the late spermatid: its central filamentous area is surrounded by chromatine particles and occasional mitochondria The immature sperm plasma membrane forms deep infoldings Mature spermatozoa from the uterus consist of a small main cell body (MCB) bearing a prominent pseudopod filled with cytoskeleton filaments The MCB contains a nucleus and mitochondria Spermatogenesis in E robusta (studied only in testes) resembles that described for P pacifica, but spermatocytes of E robusta show much lower metabolic activity and, as a result, a smaller mass of filamentous material is stored in the spermatids and immature sperm The spermatozoa of P pacifica and the immature sperm of E robusta have the main ultrastructural features characteristic for nematodes (amoeboid nature, absence of axoneme, acrosome and nuclear envelope) No aberrant organelles special for many nematode sperm (membranous organelles, paracrystalline fibrous bodies and their complexes) were found during sperm development of the chromadorids studied In this respect their spermatogenesis differs significantly from that in secernents and monhysteridsLa spermatogenese a ete etudiee en microscopie electronique a transmission chez deux especes de nematodes libres marins (Panduripharynx pacifica et Euchromadora robusta) de la famille des Chromadoridae Les spermatogonies, chez les deux especes, sont des cellules indifferenciees avec un grand noyau entoure d'une petite quantite de cytoplasme Chez P pacifica, le cytoplasme des spermatocytes contient de nombreux corps de Golgi, des cisternae du RER, des ribosomes, des mitochondries et des corps spheriques denses Le materiel filamenteux est accumule dans les spermatides qui contiennent seulement des mitochondries et un noyau fragmente (ou lobe) depourvu d'enveloppe nucleaire Le sperme immature resemble aux dernieres spermatides: son aire centrale filamenteuse est entouree par des particules de chromatine et quelques mitochondries La membrane plasmatique du sperme immature forme des invaginations profondes Les spermatozoides matures, dans l'uterus, sont constitues par un petit corps cellulaire principal (MCB) portant un pseudopode proeminent rempli de filaments de cytosquelette Le MCB contient un noyau et des mitochondries La spermatogenese chez E robusta (etudiees seulement au niveau des testicules) ressemble a celle decrite chez P pacifica, mais les spermatocytes d' E robusta sont le siege d'une activite metabolique plus faible et, par consequent, une masse plus faible de materiel filamenteux est stockee dans les spermatides et dans le sperme immature Les spermatozoides de P pacifica et le sperme immature d' E robusta ont les memes caracteristiques ultrastructurales pour des nematodes (nature amiboide, absence d'axoneme, d'acrosome et d'enveloppe nucleaire) mais aucune des organelles aberrantes particulieres a de nombreux spermes de nematodes (organelles membraneuses, corps fibreux paracrystallins et leurs complexes) n'ont ete identifiees pendant le developpement du sperme chez les Chromadorides etudies Par cet aspect, leur spermatogenese differe significativement de celle des Secernentes et des Monhysterides

Electron paramagnetic resonance spectroscopy for the investigation of the fluidity of human spermatozoa plasma membranes: a feasibility study.

Abstract: Summary. Spermatozoal membrane perturbations may play a role in abnormal sperm functions. The objective of this investigation was to study the feasibility of measuring membrane fluidity of isolated human sperm by electron paramagnetic resonance (EPR) spectroscopy and to compare the order parameter of spectra obtained from the sperm plasma membranes of living sperm of fertile men with that of infertile men. Ejaculates of infertile and fertile men were washed and the spermatozoa labelled with 5-doxylstearic acid (5-DSA) and 16-doxylstearic-acid (16-DSA) (10 nmol per 4 times 107 sperm). The reporter group of 5-DSA partitions into the outer, hydrophilic part of the sperm plasma membrane, whereas that of 16-DSA is distributed in the inner hydrophobic part. The following results were obtained: (i) the lowest measurable cell count was 3.6 to 7 times 106 sperm and the interassay variance of the orderparameter s was ≤1%; (ii) swim-up experiments revealed a higher fluidity of sperm with a higher percentage of motility; (iii) sperm membranes of infertile patients exhibited a decreased fluidity of their plasma membranes in the polar interface region of 5-DSA compared with volunteer semen donors and fertile men (P=0.002). No difference of membrane fluidity was found between the different groups using 16-DSA. It is concluded that EPR spectroscopy can be used to study the fluidity of sperm plasma membranes in fertile and infertile men.

Surface protein changes in goat spermatozoa during capacitation.

Abstract: Polypeptides of goat sperm surface before and after capacitation were examined by radiolabelling and immunologically using polyclonal antisera. Radioiodination revealed five protein bands having mol wt of 14.8, 72.4, 81, 100 and 128 kDa in uncapacitated ejaculated spermatozoa and only three bands of 23.4, 27 and 72.4 KDa in capacitated spermatozoa. The protein band with mol wt 72.4 kDa was only feebly iodinated in uncapacitated sperm surface but in capacitated spermatozoa it was heavily labelled. Western blot analysis of detergent-extracted proteins using gamma-globulin fraction of antisera raised against purified goat sperm plasma membrane revealed six antigens (17.8, 29.1, 33.4, 45.6, 85.1, 123.2 kDa) in uncapacitated spermatozoa, four (26, 32.1, 40.1, 45.6 kDa) in capacitated spermatozoa and only one (45.6 kDa) in acrosome-reacted spermatozoa. High mol wt proteins were more numerous on the surface of uncapacitated spermatozoa while the capacitated spermatozoa had relatively low mol wt proteins. An apparent effect of capacitation is the metabolism and reorganisation of proteins on goat sperm surface. Polypeptides on capacitated sperm surface revealed through radiolabelling and polyclonal antisera may have a likely receptor(s) role in the recognition and binding to homologous zona pellucida during fertilization.

Identification of homologous binding proteins in porcine and bovine gametes.

Abstract: The purpose of this study was to determine if sperm and oocyte proteins that mediate plasma membrane interaction during mammalian fertilization are conserved among porcine and bovine gametes. We examined homologous and heterologous sperm and zona-free oocyte interactions to determine the extent of cross-reactivity between the gametes of these two ungulate species. First, the numbers of ejaculated porcine and bovine sperm bound to the oocyte plasma membrane of intact porcine and bovine oocytes were determined in vitro. There was no significant difference between the number of porcine or bovine sperm that bound to porcine or bovine oocytes (P > 0.25). Second, individual porcine and bovine sperm plasma membrane proteins were identified by binding of homologous or heterologous oocyte plasma membrane to whole sperm plasma membrane on Western ligand blots. The relative amount of labeled oocyte plasma membrane bound to individual sperm plasma membrane proteins was analyzed by laser densitometry. Eight porcine sperm plasma membrane proteins and seven bovine sperm plasma membrane proteins were bound by both porcine and bovine oocyte plasma membrane. A significantly greater relative amount of porcine oocyte plasma membrane than bovine oocyte plasma membrane was bound to the 14- and 10-kD porcine sperm plasma membrane proteins (P < 0.001 and P < 0.01, respectively). A 27-kD bovine sperm plasma membrane protein bound proportionally more bovine oocyte plasma membrane probe than porcine oocyte plasma membrane probe (P < 0.04). These results are consistent with conservation of similar receptor ligand interactions at the gamete plasma membrane among porcine and bovine gametes.