Showing papers on "Sperm plasma membrane published in 2009"

PSP‐I/PSP‐II spermadhesin exert a decapacitation effect on highly extended boar spermatozoa

Abstract: PSP-I/PSP-II heterodimer is a major protein of boar seminal plasma that is able to preserve, in vitro, the viability, motility and mitochondrial activity of highly-extended boar spermatozoa. However, a relationship between the protective effects of the heterodimer and sperm capacitation is still unclear. The present study investigated the effect of the PSP-I/PSP-II (1.5 mg/mL) on membrane stability, intracellular calcium concentration ([Ca(2+)](I)) and plasma membrane and acrosome integrity of highly extended boar spermatozoa. Boar spermatozoa were diluted to 1 x 10(6) spermatozoa/mL and incubated at 38 degrees C in Phosphate-buffered saline (PBS) for 10, 30, 60, 120 and 300 min or in modified Tris-buffered medium (mTBM) for 10, 20, 30, 60 and 120 min. After each incubation time, the membrane stability (using Merocyanine-540/Yo-Pro-1), elevation of [Ca(2+)](I) (using Fluo-3-AM/PI) and the sperm plasma membrane and acrosome integrity (using SYBR-14/PI/PE-PNA) were evaluated by flow cytometry. As expected, exposure of the spermatozoa to the PSP-I/PSP-II preserved the plasma membrane and acrosome integrity compared to non-exposed spermatozoa in both media PBS and mTBM (p < .01). The evaluation of membrane stability showed no differences in the percentages of viable sperm with instable plasma membrane in the presence of the PSP-I/PSP-II compared to controls irrespective of the dilution media. The evaluation of the [Ca(2+)](I) levels showed that while spermatozoa incubated in mTBM and exposed to PSP-I/PSP-II had lower [Ca(2+)](I) than controls (39.08% vs. 47.97%, respectively; p < .05), no differences were observed in those samples incubated in PBS. However, a temporal evaluation of the samples showed that a similar proportion of live spermatozoa were able to achieve high levels of [Ca(2+)](I) and membrane instability independent of the presence of PSP-I/PSP-II. In conclusion, PSP-I/PSP-II exert a non-permanent decapacitation effect on highly extended boar spermatozoa that is related with a delay in the increase of [Ca(2+)](I) levels.

Sperm-egg adhesion and fusion in mammals.

Abstract: Fertilisation is an orchestrated, stepwise process during which the participating male and female gametes undergo irreversible changes, losing some of their structural components while contributing others to the resultant zygote. Following sperm penetration through the egg coat, the sperm plasma membrane fuses with its oocyte counterpart, the oolemma. At least two plasma membrane proteins essential for sperm-oolemma fusion--IZUMO and CD9 on the male and female gametes, respectively--have been identified recently by classical cell biology approaches and confirmed by gene deletion. Oolemma-associated tetraspanin CD81, closely related to CD9, also appears to have an essential role in fusion. Additional proteins that may have nonessential yet still facilitating roles in sperm-oolemma adhesion and fusion include oolemma-anchored integrins and oocyte-expressed retroviral envelope proteins, sperm disintegrins, and sperm-borne proteins of epididymal origin such as CRISP1 and CRISP2. This review discusses these components of the gamete fusion mechanism within the framework of gamete structure, membrane biology, cell signalling and cytoskeletal dynamics, and revisits the topic of antipolyspermy defence at the oolemma level. Harnessing the mechanisms of sperm-egg fusion is of importance to animal biotechnology and to human assisted fertilisation, wherein male patients with reduced sperm fusibility have been identified.

High Cholesterol Content and Decreased Membrane Fluidity in Human Spermatozoa Are Associated With Protein Tyrosine Phosphorylation and Functional Deficiencies

Abstract: Poor-quality sperm show reduced capacity to undergo capacitation-induced protein tyrosine phosphorylation and hyperactivation. Given that these deficiencies can be overcome by membrane-permeant stimulators of the cAMP-dependent kinase system, we hypothesize that the main defect underlying these deficiencies resides on the sperm plasma membrane. Spermatozoa from semen samples obtained from 15 consenting healthy donors were separated in 2 subpopulations, L45 (first interface) and L90 (pellet), using a 45:65:90 ISolate gradient centrifugation method. These sperm fractions were studied before and after a 6-hour capacitating incubation for sperm motion parameters (computer-assisted analysis), including hyperactivation, protein tyrosine phosphorylation (immunofluorescence), membrane fluidity (Laurdan fluorescence), and sterol and phospholipid content (high-performance thin-layer chromatography). In summary, data indicate that L45 (poor-motility) spermatozoa present an excess of cholesterol and desmosterol, which impairs the normal increase in membrane fluidity during capacitation and its consequent activation of protein tyrosine phosphorylation and hypermotility. Therefore, a defect in membrane composition and dynamics is underlying human sperm biochemical and functional deficiencies related to inadequate capacitation.

ADAM7 is associated with epididymosomes and integrated into sperm plasma membrane

Abstract: During epididymal transit, mammalian sperm acquire selected proteins secreted by the epididymis. We previously showed that a disintegrin and metalloprotease (ADAM) 7 is expressed specifically in the epididymis and transferred to the sperm surface during epididymal transit. Here, we show that mouse ADAM7 secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Furthermore, we found that ADAM7 can be transferred directly from epididymal vesicles to sperm and that it is an integral plasma membrane protein in sperm. Thus, our study provides new information regarding the unique mode of secretion and interaction of ADAM7 during the epididymis-to-sperm transfer process.

Effects of DHA-enriched hen egg yolk and L-cysteine supplementation on quality of cryopreserved boar semen.

Abstract: The objective of the present study was to determine the effects of docosahexaenoic acid (DHA)-enriched hen egg yolks and L-cysteine supplementation on the qualities of the cryopreserved boar semen. A total of 15 ejaculates from 5 Pietrain boars were divided into 4 groups according to the compositions of the freezing extenders used, that is, normal hen egg yolk (group I), DHA-enriched hen egg yolk (group II), normal hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group III) and DHA-enriched hen egg yolk with 5 mmol L(-1) of cysteine supplementation (group IV). The semen was cryopreserved using controlled rate freezer and was thawed at 50 degrees C for 12 s. Progressive motility, sperm viability, acrosome integrity and functional integrity of sperm plasma membrane of the post-thawed semen were evaluated. The supplementation of L-cysteine in the freezing extender alone (group III) improved progressive motility (P 0.05). In conclusion, the supplementation of antioxidant L-cysteine alone or in combination with DHA-enriched hen egg yolk significantly improved the post-thawed semen qualities, especially progressive motility and acrosome integrity.

Assessment of sperm quality traits in relation to fertility in boar semen.

Abstract: Background Several studies have been published where sperm plasma membrane integrity correlated to fertility. In this study we describe a simple fluorometer-based assay where we monitored the fluorescence intensity of artificially membrane-ruptured spermatozoa with a fixed time staining with fluorescent DNA dyes.

Sperm plasma membrane integrity in fertile and infertile men.

Abstract: Sperm plasma membrane characteristics were analysed by a combined method, the HOS-eosine test (HOS-E test), that consists of the hypoosmotic swelling test (HOS test), and the eosine-Y staining. Semen samples were categorized into four groups (Normo-, Oligo-, Astheno-, and Oligoasthenozoospermic) and subjected to the standard analysis (spermiogram), HOS test, eosine-nigrosine test (reflecting sperm viability); and HOS-E test. HOS-E test makes it possible to distinguish four groups of spermatozoa: type 1, HOS+/eosine-; type 2, HOS-/eosine-; type 3, HOS-/eosine+; and type 4, HOS+/eosine+. Normozoospermic samples showed 61.2 +/- 1.4% type 1, 9.2 +/- 0.8% type 2, 22.6 +/- 1.1% type 3, and 6.8 +/- 0.6% type 4 spermatozoa. Oligozoospermic samples showed no significant differences in these values, whereas asthenozoospermic samples showed a higher percentage of types 3 and 4 and a lower percentage of type 1. Oligoasthenozoospermic samples showed high percentages of types 2, 3, and 4 and a low percentage of type 1. Sperm plasma membrane integrity is a necessary condition for motility and fertilization. So, it is not surprising that semen samples with abnormal motility showed a HOS-E result indicative of a defective plasma membrane.

Severe antioxidase deficiency in human semen samples with pathological spermiogram parameters

Abstract: Spermatozoa of 103 ejaculates from infertile patients and fertile healthy individuals were separated from seminal plasma and purified on Percoll gradient to determine the activities of superoxide dismutase (SOD) and catalase (CAT) in seminal plasma as well as in spermatozoal supernatants after hypotonic disintegration of the sperm plasma membrane. Out of collected specimens, a subgroup of ejaculates from 40 individuals was examined whose female partners had developed malignant processes in the cervix uteri (oncological subgroup). All sperm samples were classified into normal and pathological semen samples according to WHO criteria. While no significant differences of SOD levels were detected in seminal plasma of patients with primary infertility, a catalase deficiency seemed to be associated with combined sperm pathology-oligoasthenoteratozoospermia (OAT). Liberated concentrations of both SOD and catalase were diminished by 10-70% in the oncological subgroup compared to normozoospermia. In four OAT samples obtained from infertile males of the oncological subgroup, total depletion from both antioxidases was observed. A lack of sufficient antioxidase protection in cases of severe sperm pathology (OAT) may also lead to cervical dysplasia.

Capacitation-associated changes in membrane fluidity in asthenozoospermic human spermatozoa

Abstract: The fertilizing potential of human spermatozoa relies on their ability to capacitate as they travel through the female reproductive tract. During this process, cholesterol is released from the plasma membrane, altering its architecture and dynamics. Using ISolate gradients, we obtained high (L90)- and low (L45)-quality spermatozoa from asthenozoospermic human semen samples. We tested the hypothesis that the lower fertilizing ability of asthenozoospermic L90 cells could be related to a lower ability to increase their membrane fluidity during capacitation. We assessed two sets of fluorescent probes: (i) DPH, TMA-DPH and PA-DPH which senses the hydrophobic core, cytosolic and exofacial leaflets of the bilayer, respectively and (ii) Laurdan, sensitive to the amount of water molecules intercalated between lipid moieties of the membrane (membrane hydration). Before capacitation, membrane fluidity of asthenozoospermic sperm populations was similar to the corresponding fractions of normozoospermic cells when evaluated with DPH, TMA-DPH or PA-DPH. Asthenozoospermic whole samples displayed lower plasma membrane hydration than normozoospermic cells as evidenced with Laurdan. After capacitation, asthenozoospermic L45 and L90 cells failed to increase their membrane fluidity in opposition to normozoospermic cells. Interestingly, membrane hydration significantly correlated with the main sperm motion parameters analysed, being a low membrane hydration associated with poor sperm movement. These results show that low-motility spermatozoa are unable to respond to capacitation with the necessary changes in membrane fluidity. This defect in sperm plasma membrane rheology may be responsible for their poor functional quality and low fertilizing ability.

Calcium requirement and time course of capacitation of goat spermatozoa assessed by chlortetracycline assay

Abstract: We standardized chlortetracycline fluorescent assay for studies of calcium requirement and time course of capacitation of goat spermatozoa Three distinct fluorescent patterns were easily detected in goat spermatozoa incubated under capacitating conditions Categorised according to nomenclature reported earlier, these are: 'F' with bright fluorescence in the postacrosomal region, characteristic of uncapacitated acrosomal-intact cells; 'B' with bright fluorescence on the anterior portion of the head and dark band in the postacrosomal region, characteristic of capacitated, acrosome-intact cells; 'AR' with lack of fluorescence on the head characteristic of acrosome-reacted cells A close correspondence was observed when the results of CTC assay were compared with those obtained by transmission electron microscopy Goat spermatozoa were not capacitated when calcium was omitted from the medium and 80% had CTC fluorescence of 'F' type The size of 'B' cell population increased with increase in calcium concentration; at 10 mmol l-1 a peak representing 65-70% capacitated cells accumulated in 4 h At higher concentrations, 'AR' cells were found along with 'B' cells and the two cell types were in equal proportions at 171 mmol l-1 Time course studies revealed a 2 h incubation period at 10 mmol l-1 and 1 h at 2 mmol l-1 calcium concentration before transformation of 'F' cells to 'B' cells was noticed However, at no time were 'AR' cells found exclusively pointing to an equilibrium between the two sperm populations Goat spermatozoa were also not capacitated when phosphate was omitted from the medium Permeant anions (NO3-, SCN-), permeant weak acid (HCO3-) and organic phosphates (beta-glycerophosphate, glucose-6-phosphate) were unable to replace phosphate The reason for their failure for the incidence of capacitation was traced to low uptake of calcium by goat spermatozoa In the presence of phosphate, a 6-8-fold increase was measured over the calcium uptake when phosphate was omitted (2-4 nmol l-1 10(8) cells-1) Mersalyl inhibited the calcium uptake by goat spermatozoa as well as its capacitation most likely by inhibiting the calcium phosphate transporter located in the sperm plasma membrane

Interspecific analysis of the glycosidases of the sperm plasma membrane in Drosophila.

Abstract: We have studied the presence of four sperm glycosidases, α-mannosidase, α-l- fucosidase and two β-hexosaminidase isoforms, in 11 species of the genus Drosophila spanning approximately an evolutionary 60 MY period, and in Scaptodrosophila lebanonensis, belonging to the ancestor genus Scaptodrosophila. These enzymes had been previously identified in Drosophila melanogaster as putative receptors for glycoconjugates of the egg surface. Alpha-mannosidase and β-hexosaminidases are intrinsic proteins of the sperm plasma membrane in species closely related to D. melanogaster as well as in the divergent species D. willistoni,D. hydei, D. virilis, and S. lebanonensis. Alpha-l-fucosidase is restricted to the species of the genus Drosophila. Alpha-mannosidase and β-hexosaminidases have been purified and characterized in all species. Their molecular masses and optimal pHs are similar in all the species, whereas interspecific differences in enzyme activities were detected. Cross-species comparison of kinetic parameters indicated a relationship between enzyme efficiency and phylogenetic relatedness. Beta-hexosaminidases were the most efficient enzymes. Lectin cytochemistry suggested the presence of carbohydrate residues complementary to the glycosidases on the eggshell at the site of sperm entry in all species. Bioinformatic analysis of the coding sequences of β-hexosaminases and α-l-fucosidase and of their predicted products showed no evidence of positive selection of the genes coding for these enzymes and a high degree of sequence identity of the predicted polypeptides among the species of the genus Drosophila. Collectively, our findings indicate that the Drosophila sperm glycosidases are structurally and functionally conserved and strengthen the hypothesis of their involvement in the interactions with the egg surface. Mol. Reprod. Dev. 76: 85–100, 2009. © 2008 Wiley-Liss, Inc.

Adhesion molecules and matrix proteins on human spermatozoa.

Abstract: Ejaculated spermatozoa and spermatogenic cells express alpha- and beta-chains of beta 1, 3 and 4 integrins as well as their ligands fibronectin and laminin in an extended intra- and interindividual variation and in different patterns of location. The mRNA transcripts of these molecules were detectable by nested polymerase chain reaction in the spermatozoa. The conclusion of a functional competence of these adhesion molecules (AM) was supported by their relation to the results of the zona-free hamster oocyte penetration (HOP) test, the in vitro fertilization of human oocytes and cell attachment assays. AM labelling was influenced by the disintegration of the sperm plasma membrane, especially in seminal plasma, by progesterone, human follicular fluid and microorganisms, but was barely modified by sperm cryopreservation. Despite substantial advances in the knowledge about sperm adhesion molecules, many questions remain to be answered.

Acaciaside-B-enriched fraction of Acacia auriculiformis is a prospective spermicide with no mutagenic property

Abstract: As a part of our continued venture to develop a safe and effective spermicide, we have identified a triterpene glycoside (Acaciaside-B (Ac-B))-enriched fraction (Ac-B-en) isolated from the seeds of Acacia auriculiformis and evaluated its spermicidal potential in vitro. Sperm motility was completely inhibited within 20 s at a minimum effective concentration (MEC) of 120 microg/ml. Tests for sperm viability by dual fluoroprobe staining showed the effect to be spermicidal with an EC(50) of 35.20 microg/ml. A series of investigations including tests for hypo-osmotic swelling, membrane lipid peroxidation, and electron microscopy document that the spermicidal effect of the fraction involves loss of sperm plasma membrane integrity and dissolution of the acrosomal vesicle--the two most important structural components that play diverse roles in physiological functions of sperm including fertilization. The fraction at 10 x MEC exerted no detrimental effects on in vitro growth of Lactobacillus acidophilus, which is considered the major constituent of vaginal microflora that maintains vaginal health. Ames tests performed with different strains of Salmonella typhimurium including TA 97a, 98, 100, and 102, which detect mutagens causing bp substitution or frameshifting at G-C or A-T bp, demonstrate no mutagenic potential of the fraction. Significant spermicidal potential with no possible mutagenic effect and adverse impacts on lactobacilli growth attests to the credential of Ac-B-en as a prospective future spermicide for the development of a safe and effective vaginal contraceptive formulation.

Testase 1 (ADAM 24) a sperm surface metalloprotease is required for normal fertility in mice.

Abstract: ADAM family members play important roles in various physiological and pathological processes, for example, fertilization, embryogenesis, neurogenesis, and development of asthma and arthritis (Primakoff and Myles, 2000. Trends Genet 16: 83-87; Edwards et al., 2008. Mol Aspects Med 29: 258-289). We previously reported that testase 1 (ADAM 24) is the first identified metalloprotease present on the surface of mature sperm. To investigate a potential role of testase 1 in fertilization, we generated testase 1 deficient mice. Testase 1 null male mice showed reduced fertility, producing only half the number of offspring when compared to wild-type littermates. In a standard in vitro fertilization assay, we found that sperm lacking testase 1 gave rise to polyspermic fertilization, a phenotypic feature that might contribute to failure of normal embryo development due to polyaneuploidy. Furthermore, in vivo, we found that testase 1 null males produced a higher number of polyspermic embryos at the pronuclear stage. These findings suggest that testase 1 is a sperm plasma membrane component which contributes to the prevention of polyspermy at the level of the oocyte plasma membrane.

Modification of Hypo-Osmotic Swelling Test to Evaluate the Integrity of Stallion Sperm Plasma Membrane

Abstract: The objectives of this study were to determine the changes in the functional integrity of the sperm plasma membrane in a total of 30 semen samples collected from five mature light breed stallions (6 each) and evaluated by using the following assays. Sperm cell progressive motility (PM), computer-aided spermatozoal analysis system (CASA), supravital stain (E, eosin-negrosin stain), standard hypo-osmotic sucrose solution (HOS, 100 mOsm), formalized hypo-osmotic sucrose solution (F-HOS, 2.85% formaldehyde) and combined standard HOS and supravital stain (HOS/E). The results revealed that, the percentage of the progressive motile spermatozoa was lower than the corresponding HOS positive percentage (HOS+%). Furthermore, the HOS+% was lower than the corresponding eosin negative percentage (E-%). Adding formaldehyde to the HOS raised the HOS+% to become close to the E-% and both positively correlated. Combined HOS test with supravital stain decreased the HOS+/E-% to the level of PM% and both positively correlated. Incubation of the sperm cells for 24 hours in formalized HOS or combined HOS with supravital staining strongly correlated to the standard HOS and with most of the conventional semen parameters. In conclusion, the modified HOS assays can improve the reliability of the HOS test in the raw stallion semen.

Protein tyrosine phosphorylation under capacitating conditions in porcine fresh spermatozoa and sperm cryopreserved with and without alpha tocopherol.

Abstract: The aim of this study was to evaluate the capacitation behaviour of fresh and alpha-tocopherol frozen spermatozoa. Spermatozoa frozen with or without alpha-tocopherol and fresh semen were incubated under capacitating conditions. Aliquots were collected at 0, 15, 30, 45, 60, 90, 120 and 180 min of incubation time. Parameters of semen quality were evaluated by optical microscopy and capacitation was determined by the epifluorescence chlortetracycline technique. Protein tyrosine phosphorylation was examined by Western immunoblotting. Motility, viability and intact spermatozoa were higher (P < 0.05) in fresh semen compared with frozen samples. These parameters significantly decreased, in every treatment, throughout the incubation time. Fresh semen showed a progressive increase in capacitated spermatozoa, reaching 25 +/- 3% at 180 min. Cryopreserved semen had a fast increase at the beginning of incubation time (28 +/- 5% at 45 min and 28 +/- 3% at 30 min for samples with or without alpha-tocopherol, respectively). The amount of an MW 32 kDa tyrosine-phosphorilated protein, associated with capacitation, increased throughout incubation for fresh semen and spermatozoa cryopreserved with alpha-tocopherol. The supplementation with alpha-tocopherol preserved sperm plasma membrane, reflected not only in the acrosome integrity but also in a greater efficiency of energy production.

Sperm plasma membrane integrity measurement: a combined method

Abstract: Sperm plasma membrane characteristics were measured by a combined method consisting of the hypo-osmotic swelling test and staining with either the eosine Y (HOS-eosine test) or propidium iodide dye (HOS-propidium test). Sperm samples were washed and resuspended in BWW medium (fraction I). Aliquots of the washed spermatozoa were treated by a swim-up technique to select motile spermatozoa (fraction II). After separation of motile cells, residual sperm pellets were treated separately (fraction III). These three fractions were subjected to the hypo-osmotic swelling test, lipid peroxidation measurement, and the HOS-eosine and HOS-propidium tests. The HOS-eosine test makes it possible to distinguish 4 types of spermatozoa: type 1: HOS+/eosine-; type 2: HOS-/eosine-; type 3: HOS-/eosine+ and type 4: HOS+/eosine+ (Fig. 1). HOS-propidium test shows equal results as HOS-eosine test. Fraction I spermatozoa showed 55.2 +/- 3.6% type 1; 12.6 +/- 1.0 type 2; 28.0 +/- 2.9 type 3; and 4.2 +/- 0.6 type 4 cells. Fraction II spermatozoa were characterized by high percentages of type 1 cells, low percentages of types 3 and 4, and very low values of lipid peroxidation (5 times smaller than fraction I). Fraction III showed a low percentage of type 1, a high percentages of the other types, and an enhanced value of lipid peroxidation (2 times higher than fraction I). The prognostic value of the HOS-eosine test was evaluated in an IVF programme. Preliminary results show that a high incidence of types 2 and 4 spermatozoa is often associated with fertilization failure.

Ghrelin enhances viability of rat spermatozoa during incubation at 37°C

Abstract: Summary Antioxidant properties of ghrelin have been demonstrated in recent studies. In the present study, the effects of chronic administration of ghrelin on the motility and plasma membrane integrity of rat spermatozoa during incubation at 37oC were investigated. Thirty 45-day-old male Wistar rats were divided into control and treatment groups. Rats in the treatment group were daily injected subcutaneously with 1 nmol of ghrelin for 10 consecutive days and the control rats received normal saline. Sperm was collected after killing of rats on days 5, 15 and 40 after the last injection, and sperm characteristics were examined at 0, 3 and 5 h after incubation at 37oC. Mass motility and forward progressive movement of spermatozoa were significantly higher in ghrelin-treated animals at 3 and 5 h of incubation on day 5 (P<0.05). After 3 h of incubation on day 15, only mass motility was greater than that of the control group. Plasma membrane integrity was assessed by hypoosmotic swelling (HOS) “water test”. The mean value of HOS reacted spermatozoa was higher in the treatment group on days 5 and 15 during 0, 3 and 5 h of incubation (P<0.05). However, the percentage of HOS-positive spermatozoa was not significantly different on day 40 between groups. There was a high correlation at 3 and 5 h of day 5 between the forward progressive movement (r = 0.92 and 0.94, P<0.0001) as well as overall sperm motility (r = 0.78 and 0.81, P<0.01) with HOS test in the ghrelin-treated animals. These results can be attributed to the antioxidative effects of ghrelin on the rat sperm especially on its plasma membrane which probably protects the sperm plasma membrane against oxidative damage during incubation and causes subsequent significant increase in the HOS test results. This may result in higher sperm motility index during 5 h of incubation.

La capacitation in vitro

Abstract: Capacitation is a prerequisite for mammalian spermatozoa to fertilize oocytes. Lipids play a crucial role in the structural and functional organization of sperm plasma membrane. Lipid and membrane protein ordering changes dramatically during sperm capacitation but the resulting effects differ according to the regions of the sperm head. Lipids modifications are mainly characterized by a cholesterol efflux, dynamic cholesterol redistribution in particular in the apical zone of the head and also a phospholipids reorganization resulting to the scramblase activation. The existence of lipids ordered microdomains (lipid rafts) has been recently observed in sperm membranes. These lipid and membrane protein movements are believed to play a role in modulating signaling pathways mainly, the AMPc/PKA and ERK pathways. One of the early key events is the activation of adenylate cyclase by high levels of bicarbonate. All these pathways lead finally to the phosphorylation of Tyr-proteins. But capacitation seems to be more complex with the contribution of other kinases (from the PI3K/Akt pathway and phosphotyrosine kinases) towards the phosphorylation of other Ser/Thr and Tyr proteins. The reactive oxygen species (ROS) seem to be important in the control of mechanisms involved in capacitation.

Protective effects of in vitro supplementation of ascorbic acid on plasma membrane, acrosomal membrane and mitochondrial activity index of human spermatozoa

Abstract: Spermatozoa were the first type of cells reported to produce free radicals. Reactive oxygen species (ROS) mediated damage to sperm is a significant contributing factor to male infertility. Impaired motility, impaired fertilization and oxidative DNA damage are three inter-related mechanisms that account for oxidative stress mediated male infertility. Spermatozoa lack cytoplasmic antioxidant defense due to exclusion of cytoplasm and therefore rely upon antioxidants present in the seminal plasma. Centrifugation of a semen sample prior to its use for intra-uterine insemination (ICI) and in vitro Fertilization (IVF) induce oxidative stress. Therefore there is need to supplement the semen with antioxidants. In the present investigation attempts were made to study the effects of in vitro supplementation of non-enzymatic antioxidant ascorbic acid on sperm plasma membrane integrity, acrosome intactness and mitochondrial activity index. There was highly significant (p<0.001) improvement in these parameters that relate to healthy state of the spermatozoa.

Possible causal factors of structural chromosome aberrations in intracytoplasmic sperm injection of the mouse

Abstract: Incidence of structural chromosome aberrations in mouse one-cell embryos produced by intracytoplasmic sperm injection (ICSI) with mature epididymal spermatozoa were influenced by sperm incubation medium and time. When spermatozoa were incubated in bicarbonate-buffered TYH for ≤0.5 h, the embryo aberration rates were significantly higher than in vitro fertilization (IVF) embryos. However, after the incubation of spermatozoa in the same medium for ≥2 h, the aberration rates were close to the IVF embryo level. When spermatozoa were incubated in bicarbonate-buffered mCZB, hepes-buffered H-TYH and H-mCZB, and phosphate-buffered PB1, the increased incidences of aberrations were observed at any incubation time. In the case of sperm incubation in H-TYH, H-mCZB and PB1, the aberration rates increased in a time-dependent manner. Chromosome aberrations generated by ICSI were transmissible to offspring. On the other hand, the aberration rate in embryos derived from testicular spermatozoa was independent of the medium type and incubation time. Thus, the incubation media appears to have no effect on sperm chromatin. TYH can effectively induce capacitation and acrosome reaction, while H-TYH, H-mCZB and PB1 never induce these spermatozoal events. It is probable that the cholesterol-rich plasma membrane and intact acrosome injected into the ooplasm affect sperm chromatin remodeling, thus resulting in the generation of chromosome damage in ICSI embryos.

Peranan antioksidan dalam pembekuan semen

Abstract: Sperm plasma membrane was rich unsaturated fatty acid and vulnerable to peroxydative damage. Vulnerability sperm against lipid peroksidation could be caused by increased cold shock. The peroxydative processed alter the structure of. sperm, particularly in the membrane and akrosom, lost motility, metabolic changed and the rapid released intraseluler component. This condition could be prevented by adding antioxidants to semen extender, such as vitamin E and BHT. Vitamin E has been proven to protected sperm plasma membrane during freezing to thawing, while Butylated Hydroxytoluene (BHT) prevent the sperm plasma membrane damage caused cold shock and provide protection against changes due to freezing. Vitamin E and BHT prevent lipid peroksidation through its the hidrogen atom to the radical peroksil rapidly. It can be concluded that an antioxidant could be prevent damage the sperm plasma membrane was caused cold sho(:k and provide protection against changes due to freezing and to prevent lipid peroksidation.

Lipids in the sperm plasma membrane and their role in fertilization

Abstract: Sexual reproduction is marked by the fusion of the sperm cell with the oocyte during fertilization to produce the diploid zygote, in which the lipids in the sperm plasma membrane play an important role. Due to the loss of most cell organelles and DNA transcription, spermatozoa lack protein expression and vesicular transport. However, the lipids of the sperm plasma membrane undergo complicated dynamic changes, which may facilitate the capacitation, binding with zona pellucida, acrosome reaction and fusion of the sperm cell with the oocyte. This paper summarizes the progress in the studies of the lipids in the sperm plasma membrane, their composition, structure, peroxidation, metabolism and role in fertilization.

Localization and expression of a 70 kDa protein in goat spermatozoa having Na + ,K + -ATPase inhibitory and arylsulphatase A activities

Abstract: We have previously isolated and purified a goat sperm protein of 70 kDa molecular weight designated as P70 and characterized it as an inhibitor of Na+,K+-ATPase. Our study reveals that the first 10 amino acid residues from the N-terminal end of P70 has high degree of homology with arylsulphatase A from mice, pig and human. Indirect immunofluorescence study shows the presence of the protein on goat sperm surface. Furthermore, live goat sperm and the extract of peripheral sperm plasma membrane proteins exhibit arylsulphatase A’s desulphation activity. The P70 remains on the head surface of in vitro capacitated cauda epididymal sperm as shown by positive immunofluorescence staining of cauda sperm. Immunoblot and flow cytometric studies corroborate the above findings. The presence of P70 on capacitated cauda sperm surface suggest a possible role of this protein in sperm zona pellucida binding. In the present report we demonstrate arylsulphatase A like activity in P70 and describe its localization and expression in goat sperm.