Showing papers on "Sperm plasma membrane published in 2004"

Role of antioxidants in treatment of male infertility: an overview of the literature

Abstract: Seminal oxidative stress in the male reproductive tract is known to result in peroxidative damage of the sperm plasma membrane and loss of its DNA integrity. Normally, a balance exists between concentrations of reactive oxygen species and antioxidant scavenging systems. One of the rational strategies to counteract the oxidative stress is to increase the scavenging capacity of seminal plasma. Numerous studies have evaluated the efficacy of antioxidants in male infertility. In this review, the results of different studies conducted have been analysed, and the evidence available to date is provided. It was found that although many clinical trials have demonstrated the beneficial effects of antioxidants in selected cases of male infertility, some studies failed to demonstrate the same benefit. The majority of the studies suffer from a lack of placebo-controlled, double-blind design, making it difficult to reach a definite conclusion. In addition, investigators have used different antioxidants in different combinations and dosages for varying durations. Pregnancy, the most relevant outcome parameter of fertility, was reported in only a few studies. Most studies failed to examine the effect of antioxidants on a specific group of infertile patients with high oxidative stress. Multicentre, double-blind studies with statistically accepted sample size are still needed to provide conclusive evidence on the benefit of antioxidants as a treatment modality for patients with male infertility.

Effect of Adding Cholesterol to Bull Sperm Membranes on Sperm Capacitation, the Acrosome Reaction, and Fertility

Abstract: When cholesterol is added to sperm membranes before cryopreservation, higher percentages of motile and viable cells are recovered after thawing. However, because one of the first steps in sperm capacitation is cholesterol efflux from the sperm plasma membrane, adding cholesterol to enhance cryosurvival may retard sperm capacitation. These studies evaluated the ability of sperm treated with cholesterol-loaded cyclodextrins (CLC) to capacitate, acrosome react, and fertilize oocytes. Control (non-CLC-treated) and CLC-treated sperm were treated with heparin, dilauroylphosphatidylcholine (PC12), or calcium ionophore A23187 (A23187) to capacitate and induce the acrosome reaction. Sperm capacitation, assessed by an increase in intracellular calcium level, and acrosome-reacted sperm were measured using flow cytometry. Fresh CLC-treated sperm cells underwent capacitation and/or the acrosome reaction at rates different from control samples, and the differences detected were dependent on the method used to induce sperm capacitation and the acrosome reaction. After cryopreservation, however, CLC-treated and control sperm underwent capacitation and the acrosome reaction at similar rates regardless of the method used to induce capacitation and the acrosome reaction. The primary concern for CLC-treated sperm, however, is whether this treatment would affect in vitro or in vivo fertility. Adding either control or CLC-treated cryopreserved sperm to bovine oocytes in vitro resulted in similar oocyte cleavage rates and blastocyst formation rates. In addition, when inseminated into heifers, pregnancy rates for control and CLC-treated sperm were also similar. Therefore, treating bull sperm with CLC permits greater numbers of sperm to survive cryopreservation while preserving the fertilizing potential of each individual sperm.

Annexin v binding and merocyanine staining fail to detect human sperm capacitation.

Abstract: The signaling pathways that characterize the process of capacitation of human spermatozoa are still largely unknown. Modifications in the lipid architecture of the sperm plasma membrane have been described in spermatozoa from different species, including translocation of phosphatidylserine (PS) from the inner to the outer leaflet and increased phospholipid disorder in the membrane. In human spermatozoa, however, results of PS exposure are controversial. In the present study, we used flow cytometry to investigate both membrane PS exposure by Annexin V (Ann V) binding and lipid disorder by merocyanine 540 (M540) staining, in swimup-selected live spermatozoa after incubation in conditions leading to capacitation. Our results indicate that neither probe is able to detect capacitation-related membrane modifications. Investigation of the nature of PS exposure and M540-positive live cells was then carried out. We found that M540 stains elements devoid of nuclei are present in seminal plasma. Live PS-exposing cells were mainly represented by damaged spermatozoa as revealed by the occurrence of a negative correlation between PS exposure and normal morphology and motility in unselected samples. The same cells were also positive for M540. These results demonstrate that Ann V and M540 binding in human sperm samples mainly detects cells with early membrane degeneration as well as dead cells, which is in agreement with findings obtained for somatic cells in which the two probes recognize cells with a damaged membrane due to the apoptotic process.

Nicotinic infertility: assessing DNA and plasma membrane integrity of human spermatozoa.

Abstract: Infertility remains a major problem in society, with recent data suggesting its presence in one of four couples. The objective of the present study was to evaluate the impact of nicotine (0.25, 0.5 and 0.75 mm), as a major component of cigarette smoke, in vitro, on sperm membrane [by spermatocrit and lipoperoxidation (LPO) tests], DNA integrity (by Comet assay), and viability of spermatozoa (by eosin staining) from normozoospermic men. Sperm samples were washed and diluted with phosphate-buffered saline. A drop in spermatocrit values and an increase in thiobarbituric acid-reactive substances/LPO rate was observed with the addition of nicotine, predominantly at a concentration of 0.75 mm, indicating a deleterious effect of nicotine on sperm membrane intactness. There was also a strong negative correlation between LPO rate and percentage viable sperm cell (r = -0.990). Data obtained from Comet assay technique revealed that nicotine could induce double-stranded DNA breaks (11% in 0.75 mm concentration) in the sperm nuclei. The value of r between LPO rate and percentage Comets was found to be +0.976. Taken together, nicotine proved to be a potential oxidant agent in the category of environmental factors to the integrity of sperm plasma membrane and DNA.

The sperm, a neuron with a tail: ‘neuronal’ receptors in mammalian sperm

Abstract: A number of plasma membrane receptor types originally thought to be specific to neurons have been found in other somatic cells. More surprisingly, the mammalian sperm and neuron appear to share many of these 'neuronal' receptors. The morphology, chromosome number, genomic activity, and functions of those two cell types are as unlike as any two cells in the body, but they both achieve their highly disparate goals with the aid of a number of the same receptors. Exocytosis in neurons and sperm is essential to the functions of these cells and is strongly influenced by similar receptors. 'Neuronal' receptor types in sperm may also play a role in the control of sperm motility (a function of course not shared by neurons). This review will consider the evidence for the presence of sperm plasma membrane 'neuronal' receptors and for their significance to mammalian sperm function. The persuasiveness of the evidence varies depending on the receptor being considered, but there is strong experimental support for the presence and importance of a number of 'neuronal' receptors in sperm.

Effects of cigarette smoking on sperm plasma membrane integrity and DNA fragmentation.

Abstract: Cigarette smoking is a serious health problem of our society. It is known that cigarette smoke is a cell mutagen and carcinogen, and that it may affect adversely male fertility. The possible detrimental effects on sperm cells are of great interest but the data available to support this statement are somewhat elusive. To approach this problem we examined conventional semen parameters, plasma membrane translocation of phosphatidylserine (PS) (annexin V/6-CFDA cell staining) and sperm DNA integrity (comet assay) in a group of healthy man smoking cigarettes on a regular basis. The results of the study were compared with the results of the same tests in healthy non-smoking donors. Significant difference in standard sperm parameters between the two groups was not found. Intensive expression of PS on the sperm plasma membrane surface (assayed by annexin V positive staining) was detected in the smokers group. There is a significant increase of population of apoptotic spermatozoa in ejaculates of smokers. Albeit DNA damages (high frequencies of double- and single- stranded DNA breaks) in spermatozoa of smokers are increased compared with non-smokers, but this difference is not statistically significant. Sperm DNA integrity of healthy smokers remains in the normal range, but a clear negative trend is observed, especially in respect of disturbance of plasma membrane phospholipid asymmetry.

Founders' Lecture. Human spermatozoa: fruits of creation, seeds of doubt.

Abstract: Deoxyribonucleic acid damage in the male germline is associated with defective fertilisation, impaired embryonic development, reduced implantation, abortion and childhood disease. Oxidative stress and the retention of excess residual cytoplasm by the spermatozoa are frequently associated with the induction of such damage. The redox cycling of xenobiotics by oxido-reductases in the germline, the patient's age, the incidence of genital tract infections and Sertoli cell dysfunction are all possible contributors to DNA damage in germ cells. Collateral peroxidation of unsaturated fatty acids in the sperm plasma membrane generally ensures that spermatozoa experiencing severe oxidative DNA damage cannot participate in the process of fertilisation. The adaptive termination of pregnancy through the selective vulnerability of genes involved in placentation may also help prevent the vertical transmission of damaged DNA. However, the ultimate safeguard against this form of damage will be to understand the biochemical basis of oxidative stress in human spermatozoa, so that the underlying causative mechanisms can be addressed in a logical manner.

Lipid diffusion in sperm plasma membranes exposed to peroxidative injury from oxygen free radicals.

Abstract: Unsaturated lipids in sperm plasma membranes are very susceptible to peroxidation when exposed to reactive oxygen species (ROS). In this investigation we have incubated ram spermatozoa in the presence of two ROS generating systems, ascorbate/FeSO4 and potassium peroxychromate (K3CrO8), and examined their effects on membrane fluidity by measuring fluorescence recovery after photobleaching (FRAP) of a lipid reporter probe 5-(N-octadecanoyl)-aminofluorescein (ODAF). Peroxidation was monitored by malonaldehyde formation and changes in fluorescence emission of 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY581/591). Ascorbate/FeSO4-induced peroxidation was inhibited by Vitamin E, butylated hydroxyanisole (BHA), 1,4-diazobicyclo(2,2,2)octane (DABCO), and to a lesser extent by ethanol. Added superoxide dismutase (SOD), gluthathione peroxidase (GPX), and catalase were ineffective scavengers. K3CrO8 induced very rapid peroxidation that could be delayed, but not prevented, by Vitamin E, BHT, DABCO, ethanol, and mannitol; once again SOD, GPX, and catalase were ineffective scavengers. Neither peroxidation with ascorbate/FeSO4 nor K3CrO8, or added H2O2 or malonaldehyde perturbed ODAF diffusion in any region of the sperm plasma membrane. Vitamin E tended to enhance diffusion rates. Exogenous cumene hydroperoxide, however, reduced ODAF diffusion to low levels on the sperm head. These results suggest that the adverse effects of ROS on spermatozoa are more likely to be caused by direct oxidation of proteins and membrane permeabilisation than disturbance of lipid fluidity. Mol. Reprod. Dev. 68: 365–372, 2004. © 2004 Wiley-Liss, Inc.

Compartmentalisation of the sperm plasma membrane: a FRAP, FLIP and SPFI analysis of putative diffusion barriers on the sperm head.

Abstract: Spermatozoa are highly polarised cells with a compartmentalised distribution of lipids and proteins in their plasma membrane. It is not known how these compartments are stably maintained in what is essentially a fluid environment. In this investigation we have examined the hypothesis that intramembranous diffusion barriers selectively retain some components within compartments, while allowing free passage of others. A fluorescence loss in photobleaching analysis of the behaviour of the lipid reporter probe 1,1′-dihexadecyl-3,3,3′3′-tetramethyindocarbocyanine (DiIC 16 ) on the head of boar spermatozoa revealed that it was freely diffusing between all three compartments (anterior acrosome, equatorial segment and postacrosome). Spermatozoa also contained rapidly diffusing particles of DiIC 16 over the anterior acrosome and equatorial segment. These particles, ∼200 nm in diameter, were tracked in real time and their trajectories analysed by mean square displacement. Particle diffusion was essentially random over the anterior acrosome and equatorial segment but showed a periodicity in jump sizes and diffusion coefficients suggestive of microheterogeneities. Particles did not exchange between the equatorial segment and postacrosome, indicating a barrier at the junction between these two compartments. No barrier was detected between the equatorial segment and anterior acrosome. A model is proposed in which a molecular `filter9 is present at the equatorial segment-postacrosomal boundary that allows free passage of single molecules but not molecular complexes. Passage of heterogeneous complexes, such as lipid rafts, requires disassembly and reassembly on either side of the filter.

Relationship Between Standard Semen Parameters, Calcium, Cholesterol Contents, and Mitochondrial Activity in Ejaculated Spermatozoa From Fertile and Infertile Males

Abstract: Purpose: To correlate levels of cholesterol (CH), calcium (Ca2+), and mitochondrial activity (MA) with the standard semen parameters and to compare them between fertile and infertile men. Methods: We studied 151 semen samples from infertile (n=60) or fertile (n=91) males. Basic sperm parameters were analyzed. Ca2+ and CH concentrations on seminal plasma were determined by enzymoimmunoanalysis. Intracellular Ca2+ and CH concentrations in the sperm plasma membrane and mitochondrial activity by fluorometry. Results: There was a significant positive correlation between sperm membrane CH and sperm morphology. Intracellular Ca2+ was lower in infertile patients compared to fertile. No differences were found regarding Ca2+ and CH concentrations in seminal plasma. MA is directly and strongly related with sperm motility. Conclusions: Intracellular concentrations of Ca2+ and the proportion of CH in the sperm membrane are two important markers of the sperm quality due to its direct relationship with sperm morphology and fertility potential.

Functional significance of the cell volume for detecting sperm membrane changes and predicting freezability in dog semen.

Abstract: Due to the similarity of plasma membrane changes induced by capacitation and cryopreservation, the parameters describing sperm response to capacitating conditions can be used for evaluating the cryopreservation response in many animal systems. In dog sperm, the response of the total sperm population to ionophore treatment has been shown to be an indication of the freezability of semen samples. Another sperm functional characteristic decisive for cryopreservability is cell volume regulation, due to the generation of essential osmotic gradients across the plasma membrane during the freeze-thaw cycles. In the present study, cryopreservation-induced changes in the membrane functional integrity were examined by monitoring the osmotically induced response of cell volume and the response to an ionophore in live cell populations. Cell volume measurements were performed on Percoll-washed suspensions of freshly diluted and frozen-thawed dog spermatozoa. The proportion of live acrosome-reacted cells was evaluated by flow cytometry after incubation under capacitating conditions in the presence of the calcium ionophore, A23187. During freezing-thawing, significant membrane changes occurred related to the disturbance of volume control ability and the loss of a proportion of live acrosome-reacted cells (P < 0.05). There were significant differences between individuals with respect to the degree of functional and structural membrane changes after thawing. Significant correlations were found between acrosomal integrity and functional membrane integrity. When assessed in freshly diluted semen, these parameters correlated with those of frozen-thawed semen samples, pointing to the similarities between mechanisms of cryopreservation-related changes and those mechanisms that mediate changes in membrane permeabilities and in cell volume regulation. The detection of changes in the sperm plasma membrane by monitoring the sperm cell volume represents a simple, rapid and sensitive method to estimate sperm quality after the cryopreservation procedure. The individual variability in response to osmotic stress or to calcium ionophore treatment appears to reflect the subtle differences in the sperm membrane functionality which are crucial for the prediction of cryopreservability.

Molecular modification of Penaeus monodon sperm in female thelycum and its consequent responses

Abstract: Using Penaeus monodon as the model, we demonstrated the molecular changes and the mechanism of thelycal-dependent sperm modification resulting in an enhanced acrosome reaction (AR) response. Attention was paid to the modification of the sperm plasma membrane which was mediated through an adsorption or removal of sperm peripheral and integral membrane proteins as indicated by the different profiles of these proteins in spermatophore (S) and thelycal (T) sperm. In vitro adsorption of Alexa-488 conjugated T proteins onto the entire S-sperm surface confirmed protein transfer in a time-dependent manner. Specific anchoring of 83 and 140 kDa proteins to sperm peripheral proteins as well as 53/55 and 60 kDa proteins to sperm lipids was demonstrated. Apart from membrane modification, a substantial increase in protein tyrosine phosphorylation was shown to be closely associated with T-dependent sperm modification event. The physiological significance of this sperm modification in enhancing sperm AR response, which required at least 3 days of T residence in order for the sperm to gain a complete AR response, was also elucidated.

Immunolocalization of cubilin, megalin, apolipoprotein J, and apolipoprotein A-I in the uterus and oviduct

Abstract: Spermatozoa maturation and capacitation occurring in the male and female reproductive tracts, respectively, involves the remodeling of the spermatozoa plasma membrane. Apolipoprotein J (apoJ) and apolipoprotein A-I (apoA-I) have been implicated in the process of lipid exchange from the spermatozoa plasma membrane to epithelial cells lining the male reproductive tract. Evidence suggests that this process is mediated by the cooperative action of the endocytic lipoprotein receptors megalin and cubilin, which are expressed at the apical surface of absorptive epithelia in various tissues, including the efferent ducts and epididymis. Here, we investigated the possibility that these receptors and their lipid-binding ligands, apoJ and apoA-I, might function similarly in the female reproductive tract. We show that megalin and cubilin are expressed in the uterine epithelium at all stages of the estrous cycle, maximally during estrous and metestrous stages. In the oviduct, there is pronounced expression of both megalin and cubilin in the nonciliated cells of the proximal oviduct and epithelial cells of the distal oviduct, particularly during estrous and metestrous stages. In both uterine and oviduct epithelial cells, megalin and cubilin were located on the apical regions of the cells, consistent with a distribution at the cell surface and in endosomes. ApoJ and apoA-I were both detected in apical regions of uterine and oviduct epithelial cells. Secretory cells of the uterine glands were found to express apoJ and apoA-I suggesting that the glands are a site of synthesis for both proteins. In summary, our findings indicate that megalin and cubilin function within the female reproductive tract, possibly mediating uterine and oviduct epithelial cell endocytosis of apoJ/apoA-I-lipid complexes and thus playing a role in lipid efflux from the sperm plasma membrane, a major initiator of capacitation.

The Contribution of d-Mannose, l-Fucose, N-Acetylglucosamine, and Selectin Residues on the Binding of Glycodelin Isoforms to Human Spermatozoa

Abstract: Previous data showed that glycodelin-A from amniotic fluid and glycodelin-F from follicular fluid inhibited sperm-zona pellucida binding. Solubilized zona pellucida reduced the binding of glycodelin-F to sperm extract dose dependently. This study demonstrated that the zona pellucida proteins also reduced the binding of glycodelin-A to sperm extract. Ionophore-induced acrosome reaction reduced the binding of iodinated glycodelin-A and -F to sperm, indicating that the glycodelin-binding sites are on the outer acrosomal membrane or on the sperm plasma membrane overlying the acrosome. While the binding of glycodelin-A to sperm was suppressed by mannose and fucose neoglycoproteins, that of glycodelin-F was also reduced by acetylglucosamine neoglycoprotein. Pretreatment of sperm with inhibitors of mannosidase and acetylglucosaminidase reduced the binding of glycodelin-F to sperm. On the other hand, inhibitor of mannosidase but not of acetylglucosaminidase inhibited the binding of glycodelin-A. In a competition binding assay, mannosidase reduced both glycodelin-A and -F binding whereas acetylglucosaminidase reduced only glycodelin-F binding. While fucosidase reduced the binding of both glycodelins, fucosidase inhibitor was marginally active in suppressing the binding of glycodelins to human sperm. Among the selectins tested, only E-selectin had a slight inhibitory effect on the binding of glycodelin-A to sperm. The binding of glycodelin-F was unaffected by selectins and their antibodies. In conclusion, the binding of glycodelin-A to sperm involves mannose, fucose, and possibly E- selectin residues, while that of glycodelin-F involves mannose, fucose, and N-acetylglucosamine but not the selectin residue.

Antisperm antibodies modify plasma membrane functional integrity and inhibit osmosensitive calcium influx in human sperm

Abstract: BACKGROUND: The hypo-osmotic swelling (HOS) test evaluates the ability of the functional sperm plasma membrane to stretch following cell swelling when exposed to hypo-osmotic solutions. Sperm samples with low HOS scores show low fertilization and pregnancy rates during assisted reproductive techniques, though data are controversial. The aim of this study was to compare the results of the HOS test in a group of normozoospermic men with those in a group of subjects affected by autoimmune infertility due to the presence of antisperm antibodies (ASA) bound to the sperm surface. METHODS: Sperm from normozoospermic and from infertile subjects affected by autoimmune infertility were exposed to hypo-osmolar conditions to verify the effects on intracellular calcium concentrations and acrosome reaction. RESULTS: Sperm samples from infertile men with ASA showed HOS test scores that were significantly lower than those of normozoospermic subjects despite similar sperm viability percentages. Sperm with ASA bound to their plasma membrane showed a reduced rise in intracellular calcium concentrations and acrosome reaction after hypo-osmotic challenge with respect to sperm from normozoospermic subjects without ASA. CONCLUSIONS: Infertile subjects with ASA have a reduced sperm plasma membrane functional integrity that could explain, at least in part, the low fertilization and pregnancy rates observed in these subjects during assisted reproductive procedures. Evaluation for the presence of ASA in all sperm samples showing low HOS test scores in the presence of normal sperm viability percentages is suggested.

002. Human spermatozoa: fruits of creation, seed of doubt

Abstract: Defective sperm function is the largest defined cause of human infertility, affecting one in twenty Australian males. Despite its prevalence, we are only just beginning to understand the underlying mechanisms. The past decade has seen two major advances in this field: (1) the discovery that Y chromosome deletions play a key role in the aetiology of non-obstructive azoospermia/oligozoospermia; and (2) recognition that oxidative stress can impact upon the functional competence of human spermatozoa through peroxidative damage to the sperm plasma membrane. Oxidative stress has also been found to disrupt the integrity of DNA in the male germ line and may represent an important mechanism by which environmental impacts on human health are mediated. Thus, paternal exposure to various toxicants (cigarette smoke, organic solvents, heavy metals) has been linked with oxidative DNA damage in spermatozoa and developmental defects, including cancer, in the F1 generation. The male germ line becomes particularly vulnerable to such factors during the post meiotic stages of differentiation. Pre-meiotic germ cells always have the option of undergoing apoptosis if DNA damage is severe. However, post meiotic germ cells have lost both the ability to mount an apoptotic response and the capacity for DNA repair. As a result, germ cells are particularly vulnerable to genotoxic agents during spermiogenesis and epididymal maturation. If the fertilizing capacity of the spermatozoa is retained following toxicant exposure, then DNA damage will be transferred to the zygote and must be repaired subsequently by the oocyte and/or early embryo. Aberrant DNA repair at this stage has the potential to create mutations that will compromise embryonic development and, ultimately, the normality of the offspring. Elucidating the causes of oxidative damage in spermatozoa should help resolve the aetiology of conditions such as male infertility, early pregnancy loss and childhood disease, including cancer.

A third sea urchin sperm receptor for egg jelly module protein, suREJ2, concentrates in the plasma membrane over the sperm mitochondrion.

Abstract: Sea urchin spermatozoa are model cells for studying signal transduction events underlying flagellar motility and the acrosome reaction. We previously described the sea urchin sperm receptor for egg jelly 1 (suREJ1) which consists of 1450 amino acids, has one transmembrane segment and binds to the fucose sulfate polymer of egg jelly to induce the sperm acrosome reaction. We also cloned suREJ3 which consists of 2681 amino acids and has 11 putative transmembrane segments. Both these proteins localize to the plasma membrane over the acrosomal vesicle. While cloning suREJ1, we found suREJ2, which consists of 1472 amino acids, has two transmembrane segments and is present in the entire sperm plasma membrane, but is concentrated over the sperm mitochondrion. Experimental evidence suggests that, unlike suREJ1 and suREJ3, suREJ2 does not project extracellularly from the plasma membrane, but is an intracellular plasma membrane protein. All three sea urchin sperm REJ proteins possess a protein module of > 900 amino acids, termed ‘the REJ module’, that is shared by the human autosomal dominant polycystic kidney disease protein, polycystin-1, and PKDREJ, a testis-specific protein in mammals whose function is unknown. In the present study, we describe the sequence, domain structure and localization of suREJ2 and speculate on its possible function.

Effect of Exogenous Progesterone on Post-thaw Capacitation and Acrosome Reaction of Bovine Spermatozoa

Abstract: The aim of this work was to study the effect of progesterone (P4) on capacitation and acrosome reaction (AR) of post-thaw bovine spermatozoa in vitro. Spermatozoa were incubated (0-180 min) in capacitation medium supplemented with 0, 0.1, 1.0 and 10.0 microg/ml of P4. At different time intervals aliquots were taken to determine sperm plasma membrane lipid destabilization, or capacitation (AR induced by lysophosphatidylcholine) in spermatozoa. The second experiment aimed to study the effects of P4, as potential inducer of AR in heparin-capacitated spermatozoa. The acrosomal status and viability of spermatozoa were evaluated under an epifluorescence microscope using Ethidium homodimer/peanut agglutinin fluorescein isothiocyanate staining method. Plasma membrane scrambling in spermatozoa was assessed by a flow cytometer, using merocyanine staining. The results show that P4 at the concentrations used had no negative effects on sperm viability. Progesterone significantly enhanced sperm capacitation (p 0.05) and did not significantly increase the AR of heparin-capacitated spermatozoa (p > 0.05). Progesterone displayed its effects in a dose-dependent manner with a maximum effect of 10 microg/ml P4 at 180 min of incubation. The results demonstrate that in cryopreserved bovine semen, P4 acts as capacitating, but not as an AR-inducing agent.

Measuring ion fluxes in sperm.

Abstract: Publisher Summary The chapter discusses different strategies to study ion fluxes in sperm. Ion channels and transporters in the sperm plasma membrane participate crucially in fertilization. Ion-permeability changes are deeply involved in how sperm sense environmental cues and signals from the outer envelope of the egg to achieve fertilization. Combining the in vivo measurements of intracellular ions and membrane potential in sperm populations and single cells with electrophysiological approaches, such as the smart patch-clamp, and reconstitution strategies in planar bilayers reveal how sperm ion channels participate in sperm motility and the acrosome reaction. This chapter describes ion transport protocols for sea urchin sperm. Egg ligands immediately induce sperm responses. It is important to measure the rapid kinetics of their responses to understand the underlying signaling mechanisms. To perform a successful time-resolved measurement, it is essential that sperm be exposed to egg ligands as rapidly as possible. There are two different methods to achieve this: (1) rapid mixing (stopped-flow fluorometry) and (2) photolysis of caged (photoactivatable) ligands. The chapter presents techniques used in the study of ion fluxes in single sea urchin sperm.

208 effect of refrigeration and cryopreservation on the quality of lion epididymal spermatozoa

Abstract: Epididymal spermatozoa from harvested wild animals is potentially useful for conservation purposes, as it can be used for subsequent artificial insemination or stored in Biological Resource Banks for future use. The potential of sperm banking is of particular interest for use in lion (Panthera leo) populations maintained in small National Parks, as translocation of males to effect gene-flow is often problematic, resulting in the translocated lion being killed by resident pride males. We measured the change in sperm quality over time during cool storage (at 4°C) and after thawing of samples cryopreserved at −196°C. Also, we present a correlation between sperm plasma membrane integrity and mitochondrial activity as measured by fluorescent analysis. The testes from a pride lion were removed and transported to the laboratory (at 4°C) within 6 h. The epididymides were removed and both cauda epididymides were flushed with 1 mL of Tris-citrate egg yolk extender (Fraction A, Biladyl, Minitub, Germany). The sample containing 2930 × 106 cells mL−1 was washed (20 mM HEPES, 355 mM sucrose, 10 mM glucose, 2.5 mM KOH;; 400 mOsm/kg, pH 7; Sigma, South Africa) and after centrifugation (5 min. at 600g), the pellet was resuspended in 0.5 mL of washing solution (with 197 mM NaCl instead of sucrose). One aliquot of spermatozoa was kept at 4°C and evaluated at 24 h intervals for 7 days. A second aliquot of the sperm sample was extended in Tris-citrate egg yolk extender with glycerol (Fraction B, Biladyl), frozen in liquid nitrogen (LN) vapor and stored in LN. The frozen sample was later thawed and evaluated as for the cooled samples. Percentages of motile (MS) and progressive (PS) spermatozoa were assessed using a phase contrast microscope (×200; stage at 37°C). Sperm plasma membrane damage was assessed by determining the percentage of cells exhibiting red fluoresence after staining with propidium iodide (PI, 50 ng/mL; 10 min RT). Spermatozoa that did not stain red in PI were classified as plasma membrane intact (PMI). Resilience to hypo-osmotic shock and plasma membrane integrity were evaluated by incubating a portion of the sample in a 100 mOsm/kg solution (10 nM glucose, 20 nM HEPES, 30 nM NaCl) containing PI for 15 min at room temperature. The percentage of sperm cells with active mitochondria (MIT) was determined by counting spermatozoa showing orange fluoresence over the mid-piece after staining with JC-1(7.5 uM Sigma) for 30 min at 37°C. At collection, MS was 15% and did not show a significant decrease during the 7-day storage period. Initially, PS was 10% and dropped to 5% after 7 days, with values fluctuating during the storage period. Both PMI and HOSPMI were 80% on Day 1, gradually decreasing to 75% on Day 7 of storage. PMI and MIT showed a highly significant correlation (r = 0.88; P = 0.003; n = 8). In frozen-thawed sperm samples, MS fell from a pre-freeze value of 15% to 5% after thawing. Similarly, PS fell from 10% in pre-freeze to 3% in frozen-thawed samples. Likewise, PMI, HOSPMI and MIT values were 80% and 45%, 87% and 45% and 89% and 49%, respectively. Our study showed that lion sperm PMI and MIT remained high after 7 days at 4°C. MS and PS, although low, did not vary during this same period. PI and JC-1 showed a significant correlation, suggesting that both might be affected by the same deleterious factors. Although PMI, HOSPMI and MIT values decreased approximately 40% after freezing, we feel that such sperm samples could be used for in vitro embryo production, if not by IVF, by ICSI. Of course, additional studies are needed to validate our suggestion.

Cryoprotective Effect of Four Penetrating Cryopro-tectants on Rhesus Monkey (Macaca mulatta) Spermatozoa Cryopreservation

Abstract: The present study aimed to examine four cryoprotectants:glycerol,dimethyl sulfoxide,ethylene glycol and propylene glycol on rhesus monkey sperm cryopreservation.Frozen-thawed sperm cryo-survival and function was evaluated by sperm motility,plasma membrane integrity and acrosome integrity,and sperm plasma membrane and acrosome status was determined by nuclei stain Hoechst 33258 and Fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA).The result showed that:for the ability of protection on post-thaw sperm motility,glycerol (47.3±5.7%) and ethylene glycol (44.8±6.7%)> dimethyl sulfoxide (22.9±0.9%)> ethylene glycol (0±0%);for the ability of protection on sperm plasma membrane integrity,glycerol (54.8±3.2%) and ethylene glycol (54.0±6.7%)> dimethyl sulfoxide (37.5±7.0%)> ethylene glycol (28.3±6.5%);for the ability of protection on sperm acrosome integrity,glycerol (82.2±2.4%) and ethylene glycol (82.4±2.4%)> dimethyl sulfoxide (68.7±5.7%) and ethylene glycol (72.3±3.5%) (P<0.05).The results indicate that cryoprotective effects of various penetrating cryoprotectants are different.Dimethyl sulfoxide and propylene glycol are not suitable for rhesus monkey sperm freezing,especially propylene glycol.Ethylene glycol with similar cryoprotective properties to glycerol could be successfully used in the cryopreservation of rhesus monkey spermatozoa.

EFECTO DE LA LISOFOSFATIDILCOLINA EN LA REACCIÓN DEL ACROSOMA DE ESPERMATOZOIDES CANINOS Effect of Lysophosphatidylcholine on Acrosome Reaction in Canine Spermatozoa

Abstract: Lysophospholipids desestabilize the sperm plasma membrane and promote its fusion with outer acrosomal membranes, by accelerating acrosome reaction (RA). Lysophosphatidylcholine (LC) has been used to induce the RA on capacitated sperms from different mammals. The aim of this work was to evaluate the effect of LC on RA in canine spermatozoa. Different concentrations of LC (0, 100, 200 and 300 µg/mL) were utilized during 15 minutes to induce RA in spermatozoa incubated during 0, 3 and 4 hours in capacitation medium (mCCM). Sperm viability and acrosomal status were determined using double fluorescence Pisum sativum aglutinin with fluorescein isothiocyanate (PSA-FITC) and Hoechst 33258 The analysis of varience (ANOVA) test was utilized for statistical analysis. The sperm viability was significantly reduced with 200 and 300 µg/mL of LC (P 0.05) between groups with 0 and 100 µg/ml of LC incubated to 0 hour (21.0 ± 4.2% vs. 21.0 ± 6.6%), 3 hours (43.8 ± 4.7% vs. 49.1 ± 5.2%) and 4 hours (51.3 ± 14.8% vs. 57.6 ± 9.9%). Nevertheless the percentage of live spermatozoa with RA increased (P<0.05) with incubation at 3 and 4 hours. In conclusion, LC (100 µg/mL) shows no significant effect for inducing the acrosome reaction in canine spermatozoa incubated in mCCM mediun free of glucose.