Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll^® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.     

Lipid composition and metabolism in testicular and ejaculated ram spermatozoa

1. Spermatozoa collected directly from the testis of the conscious ram contain 25% more phospholipid than ejaculated spermatozoa. The concentration of lecithin, phosphatidylethanolamine and ethanolamine plasmalogen was greater in testicular spermatozoa; little difference was observed in choline plasmalogen. Both types of spermatozoa had significant amounts of cardiolipin and alkyl ether phospholipid. 2. The fatty acids in the phospholipid extracted from testicular spermatozoa have a very high content of palmitic acid. The phospholipids of ejaculated spermatozoa contained less palmitic acid, but more myristic acid. 3. Ejaculated spermatozoa contained less acyl ester and cholesterol. It is suggested that lipids are a source of substrate for spermatozoa during their passage through the epididymis. 4. Testicular spermatozoa when incubated with [U-¹⁴C]glucose incorporated more radioactivity into the glycerol part of the phospholipid and neutral lipid fractions than did ejaculated cells. The distribution of radioactivity in the individual phospholipids and neutral lipids was similar for both cell types. No radioactivity was detected in choline plasmalogen, which accounted for approx. 40% of the total phospholipid. 5. Testicular spermatozoa incorporated more radioactivity from glucose into formate than into acetate, whereas a higher proportion of radioactivity was found in acetate in ejaculated cells. 6. The implications of these lipid changes in the process of spermatozoal maturation are discussed.     

Composition lipidique des spermatozoides humains et susceptibilité au stress oxydant avant et après migration dans le mucus cervical

Spermatozoa are particularly susceptible to damage induced by ROS, especially as their plasma membrane contains large amounts of polyunsaturated fatty acids. Mammalian sperm cells develop the capacity to fertilise ova during transport in the male and female reproductive tracts. The nature and quality of the micro-environment of the female reproductive tract are important factors for sperm selection, capacitation and subsequent acrosome reaction.In vitro experiments using capacitating media have shown remodeling of the lipid composition of the sperm membrane during these steps and the same approaches have also shown that a low level of ROS was necessary. The oxidative status of the female genital tract is therefore certainly of primary importance for the physiological maturation of the sperm cell. It has been previously reported that an inappropriate oxidative balance in the male genital tract (ie, an excessive ROS production overwhelming all antioxidant strategies) impairs the structure and several functions of sperm cells. This phenomenon may arise in the female genital tract, but has never been investigated. The present paper is a review of the literature on these subjects and also reports our results concerning the changes in semen lipid content during cervical mucus migration and the effect of cervical mucus polymorphonuclear (PMN) cells on sperm characteristics. We showed that the sperm levels of vitamin E, cholesterol, phospholipids, sphingomyelin and plasmalogen assessed by HPLC decreased after migration through cervical mucus. These modifications were observed in parallel with lipid enrichment of the cervical mucus, suggesting an efflux of cholesterol and lipids from sperm cells. The spermatozoa recovered postmigration in the cervical mucus were characterised by low levels of the various lipid classes. Spermatozoa that migrated in cervical mucus samples with a considerable quantity of polymorphonuclear leukocytes (PMN) also showed significantly increased levels of sphingomyelin, diacyl phospholipids and plasmalogens in comparison to spermatozoa that migrated in cervical mucus devoid of PMN. Finally, we also found that PMA-induced ROS production was significantly increased for spermatozoa treated with cervical mucus containing PMN.     

Glutathione-S-transferase: Role in buffalo (Bubalus bubalis) sperm capacitation and cryopreservation

In this study, glutathione-S-transferase Mu3 (GST) has been reported to play an important role in sperm capacitation, acrosome reaction, and fertilization. The freshly ejaculated buffalo spermatozoa were in vitro capacitated using heparin (10 μg/mL) or cryopreserved in egg yolk citrate extender. Glutathione-S-transferase was identified and characterized in terms of their isozymic forms, tyrosine phosphorylation, and immunolocalization patterns in cryopreserved buffalo spermatozoa in comparison with freshly ejaculated and in vitro capacitated spermatozoa. Two-dimensional gel electrophoresis, immunoblot, immunocytochemistry, and enzyme activity analyses were done to characterize GST in this study. Five and eight isozymic forms of GST were detected in cryopreserved and capacitated spermatozoa, respectively. Differential tyrosine phosphorylation of these enzymes was observed in cryopreserved and capacitated spermatozoa. The tyrosine phosphorylation of this enzyme involved cAMP protein kinase-A dependent and extracellular signal-regulated kinase independent pathways during in vitro capacitation of the spermatozoa. Differential immunolocalization patterns of GST were observed in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Glutathione-S-transferase Mu3 enzyme activity was found to be significantly (P < 0.05) different in freshly ejaculated, capacitated, and cryopreserved spermatozoa. Activity of GST was significantly (P < 0.05) increased with the progression of capacitation. The cryopreserved spermatozoa showed significantly (P < 0.05) greater enzyme activity compared with fresh spermatozoa and was equal to 2-hour capacitated spermatozoa. The cryopreserved spermatozoa showed significant (P < 0.05) loss of GST enzyme protein. Tyrosine phosphorylated GST showed significantly (P < 0.05) greater activity compared with their dephosphorylated forms. The information generated in this study can be used to understand the molecular mechanism of the effects of GST on capacitation. Regulation of GST during sperm cryopreservation could be a good target to improve fertility of cryopreserved spermatozoa for their use in assisted reproductive technologies.     

Quality and lipid composition of spermatozoa in rabbits fed DHA and vitamin E rich diets

The effects of fish oil (FO) and vitamin E (vE) dietary supplementation on semen quality, sperm susceptibility to lipid peroxidation, tocopherols content and fatty acid profiles were studied in rabbits. Fifty-two rabbit bucks randomly divided in four groups received a control diet and enriched diets containing either FO (1.5%, w/w), vE (200 mg/kg) or both. Semen volume, concentration, motility and viability were analysed at various time-points and the lipid composition was assessed on sperm cells. The phospholipid fatty acid profile was determined: n-6 PUFA were the major fatty acids found, with a proportion of 42%, whereas the n-3 PUFA accounted for nearly 1%, mainly represented by C22:6n-3 (docosahexaenoic acid, DHA). FO supplementation produced a seven-fold increase in the content of DHA in sperm phospholipids and a comprehensive rearrangement of the phospholipid fatty acid composition, while an unexpected negative effect of feeding high level of vE on the proportion of total PUFA was found. Despite the remarkable changes observed in sperm lipid composition, semen quality parameters were not affected by the dietary treatments and the interaction between the two dietary supplements had a significant effect only on sperm concentration. An increase in semen production by ageing and a concomitant rise in sperm susceptibility to in vitro peroxidation was found. α- and δ-tocopherol, present in rabbit sperm in similar amount, were not affected by dietary treatment. δ-tocopherol content had a significant linear negative regression with age and showed a significant negative correlation with the susceptibility to peroxidation values.     

Studies on the mechanism of capacitation II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro

1. 1. Evidence has been provided for the transfer of phosphatidyl[¹⁴C]choline and [³H]cholesterol between bovine serum albumin and cauda epididymal rat spermatozoa in Krebs-Ringer bicarbonate medium, which can promote sperm capacitation.

2. 2. An analysis of the lipid composition in both albumin and spermatozoa revealed that phospholipid levels decreased in the protein and increased by roughly comparable amounts in sperm cells during incubation in vitro.

3. 3. Cholesterol (free + ester) increased in albumin and decreased in spermatozoa. Changes in the amount of esterified cholesterol were solely responsible for the increase associated with albumin, whereas whole sperm cell extracts showed a significant decline in free cholesterol.

4. 4. The composition of albumin-bound fatty acids did not alter appreciably as a result of incubation with spermatozoa.

5. 5. Rates of [¹⁴C]palmitic acid utilization by spermatozoa suggest that lipid synthesis accounted for less than 5% of the changes observed under the conditions of this study.

6. 6. These results are interpreted as broadly supporting our previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro. Specifically, the results are compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.

Vesicular transfer of membrane components to bovine epididymal spermatozoa

Epididymosomes (apocrine secreted epididymal vesicles) are assumed to play a crucial role in sperm maturation. Our aim has been to analyze the fusogenic properties of bovine epididymosomes and their involvement in the transfer of membrane components (lipids, proteins, plasma membrane Ca²⁺-ATPase 4 [PMCA4]) into bovine sperm. The fusogenic properties of epididymosomes with spermatozoa were investigated in vitro by using octadecyl rhodamine-B (R18)-labeled epididymosomes. Spermatozoa isolated from the epididymal caput showed a higher fusion rate than those taken from the cauda. The fusion rate was dependent on pH and time. Furthermore, the lipid and protein content in spermatozoa changed during epididymal transit and after in vitro fusion with epididymosomes. Following the in vitro fusion of caput spermatozoa with epididymosomes, the cholesterol/total phospholipid ratio of the sperm plasma membrane decreased. The effect was comparable with the cholesterol/total phospholipid ratio of native cauda spermatozoa. Co-incubation experiments of spermatozoa with biotinylated epididymosomes additionally revealed that proteins were transferred from epididymosomes to sperm. To examine the potential transfer of epididymis-derived PMCA4 to spermatozoa, immunofluorescence analysis and Ca²⁺-ATPase activity assays were performed. In caput spermatozoa, the PMCA4 fluorescence signal was slightly raised and Ca²⁺-ATPase activity increased after in vitro fusion. Thus, our experiments indicate significant changes in the lipid and protein composition of epididymal sperm following interaction with epididymosomes. Moreover, our results substantiate the presumption that PMCA4 is transferred to spermatozoa via epididymosomes.     

Full-term development of rats from oocytes fertilized in vitro using cryopreserved ejaculated sperm

For preservation of rat spermatozoa, the general-purpose method requires that the male be sacrificed for collection of spermatozoa from the epididymides. However, it would be highly useful if the ejaculated spermatozoa could be successfully cryopreserved and the frozen–thawed spermatozoa used for in vitro fertilization, since this would allow the genetically valuable rats to be maintained alive rather than sacrificed. The aim of the present study was to clarify whether ejaculated rat spermatozoa could be successfully cryopreserved and fertilized in vitro. The motility and viability of frozen–thawed ejaculated spermatozoa were similar to those of frozen–thawed epididymal spermatozoa (around 10%). The percentage of acrosomal integrity in epididymal spermatozoa was significantly higher than that in ejaculated spermatozoa after freezing/thawing. The level of capacitation-associated protein tyrosine phosphorylation in frozen–thawed ejaculated sperm was slightly increased at 5 h. When the frozen–thawed ejaculated spermatozoa were used for in vitro fertilization, the percentages of fertilization, pronuclear formation, and development to the 2-cell stage (26.5%, 23.0%, and 91.0%, respectively) were similar to those of frozen–thawed epididymal spermatozoa (19.4%, 15.0%, and 84.1%, respectively). However, the rate of blastocyst formation in the ejaculated group was significantly lower than that in the epididymal group (12.0% vs 43.2%). Results from the embryo transfer experiment showed that the proportions of embryos developed to term were similar between the ejaculated (47.7%) and epididymal groups (53.7%). We showed here for the first time that ejaculated spermatozoa can be cryopreserved and the frozen–thawed sperm could be developed to term via in vitro fertilization in rats.     

Spermatogenesis of Zaprionus indianus and Zaprionus sepsoides (Diptera,Drosophilidae): Cytochemical,structural and ultrastructural characterization

Zaprionus indianus is a drosophilid native to the Afrotropical region that has colonized South America and exhibits a wide geographical distribution. In contrast, Z. sepsoides is restricted to certain African regions. The two species differ in the size of their testes, which are larger in Z. indianus than in Z. sepsoides. To better understand the biology and the degree of differentiation of these species, the current study evaluated spermatogenesis in males of different ages by conventional staining techniques and ultrastructural analysis. Spermatogenesis and the ultrastructure of spermatozoa were similar in the two species, and the diploid number was confirmed to be 2n = 12. A greater number of spermatozoa were observed in young Z. indianus (1–3 days old) compared to Z. sepsoides males, which showed a higher frequency of cells at the early stages of spermatogenesis. The head of the sperm was strongly marked by silver staining, lacto-acetic orcein and the Feulgen reaction; the P.A.S. reaction revealed glycogen granules in the testes of both species. Both species presented similar arrangement of microtubules (9+9+2), two mitochondrial derivatives of different size and 64 spermatozoa per bundle. Such similarity within the genus Zaprionus with other species of Drosophila, indicates that these structures are conserved in the family Drosophilidae. The differences observed the number and frequency of sperm cells in the early stages of spermatogenesis, between the young males of Z. indianus and Z. sepsoides, are features that may interfere with reproductive success and be related to the invasive potential of Z. indianus.     

Characterization of cAMP-dependent protein kinase and its endogenous substrate proteins in ram testicular,cauda epididymal,and ejaculated spermatozoa

Mammalian spermatozoa have been shown to possess cAMP-dependent protein kinase (A-PK) and endogenous substrate proteins for this enzyme. A study of the kinase system was undertaken to determine changes that may be associated with sperm maturation by comparing immature testicular with mature cauda epididymal and ejaculated spermatozoa. Absolute activity levels of A-PK, stimulated over a concentration range of 10^?9 to 10^?5 M, was significantly greater in testicular than ejaculated spermatozoa. At an optimal cAMP concentration (10^?6M), testicular spermatozoa had significantly greater amounts of cAMP-dependent protein kinase activity than did cauda or ejaculated spermatozoa. Electrophoretic analysis and autoradiography of NP-40-soluble protein extracts revealed the presence of two substrate proteins (M_r = 62,000 and 44,000) in all three types of spermatozoa. In addition, a phosphoprotein (M_r = 20,000) was detected in mature cauda and ejaculated but not immature testicular spermatozoa. The phosphorylation of these substrate proteins was both dose and time dependent. Examination of cyclic AMP phosphodiesterase activity revealed significantly higher levels in testicular than ejaculated spermatozoa. These results indicate marked alterations in cAMP-modulated protein phosphorylation and dephosphorylation systems in ram spermatozoa during epididymal maturation.     

Carbon and hydrogen isotopic fractionation during lipid biosynthesis in a higher plant (Cryptomeria japonica)

Compound-specific carbon and hydrogen isotopic compositions of lipid biomolecules (n-alkanes, n-alkanoic acids, n-alkanols, sesquiterpenes, diterpenes, phytol, diterpenols and β-sitosterol), extracted from Cryptomeria japonica leaves, were determined in order to understand isotopic fractionations occurring during lipid biosynthesis in this species. All lipid biomolecules were depleted in both ¹³C and D relative to bulk tissue and ambient water, respectively. n-Alkyl lipids associated with the acetogenic pathway were depleted in ¹³C relative to bulk tissue by 2.4-9.9‰ and depleted in D relative to ambient water by 91-152‰. C₁₅- and C₃₀-isoprenoid lipids (sesquiterpenes, squalene and β-sitosterol) associated with the mevalonic-acid pathway are depleted in ¹³C relative to bulk tissue by 1.7-3.1‰ and depleted in D relative to ambient water by 212-238‰. C₂₀-isoprenoid lipids (phytol and diterpenoids) associated with the non-mevalonic-acid pathway were depleted in ¹³C relative to bulk tissue by 4.6-5.9‰ and depleted in D relative to ambient water by 238-303‰. Phytol was significantly depleted in D by amounts up to 65‰ relative to other C₂₀ isoprenoid lipids. The acetogenic, mevalonic-acid and non-mevalonic-acid pathways were clearly discriminated using a cross-plot between the carbon and hydrogen isotopic fractionations.     

The yeast O-acyltransferase Gup1p interferes in lipid metabolism with direct consequences on the sphingolipid-sterol-ordered domains integrity/assembly

Saccharomyces cerevisiae Gup1p is a membrane-bound O-acyltransferase. Previous works involved GUP1 in a wide range of crucial processes for cell preservation and functioning. These include cytoskeleton polarization and secretory/endocytic pathway, GPI-anchor remodelling, wall composition and integrity, and membrane lipids, with a reduction in phospholipids and an increase in acylglycerols. DRM fractions were found in considerably lower amounts in gup1Δ than in wt strain. Additionally, the proteins presumably associated with lipid micro domains, Gas1p and Pma1p, were present in much smaller amounts in the mutant DRMs. Pma1p is also found in minor quantities in the whole cells extracts of the gup1Δ mutant. Accordingly, H⁺-ATPase activity was reduced in about 40%. Deletion of GUP1 resulted in higher sensibility to specific sphingolipid biosynthesis inhibitors and a notorious resistance to ergosterol biosynthesis inhibitors. Furthermore, the majority of mutant cells displayed an even (less punctuated) sterol distribution. The present work presents improvements to DRMs extraction methodology and filipin-sterol staining, provides evidence supporting that Gup1p is involved in lipid metabolism and shows the direct consequences of its absence on the plasma membrane sphingolipid-sterol-ordered domains integrity/assembly.     

Protective effects of propolis on cryopreservation of common carp (Cyprinus carpio) sperm

Cryopreservation of sperm is common procedures in aquaculture, particularly used for routine in artificial insemination. However, these application cause damages and adversely affected sperm motility, viability and consequently lower hatching rates. The objective of this study is to determine whether propolis has an effect on cryopreservation and fertilization ability and to investigate the potential protective effect of propolis on spermatozoa of Cyprinus carpio. Many studies have been done in cryopreservation offish spermatozoa, but none of them contain propolis in extender composition. The extenders were prepared by using modified Kurokura Solution to which 10% Me₂SO added with different levels of propolis (0.2, 0.4, 0.6, 0.8 and 1 mg ml⁻¹) and 10% egg yolk (as a control without propolis). The pooled semen samples diluted at the ratio of 1:9 by the extenders were subjected to cryopreservation. The percentage and duration of motility and fertilization tests of cryopreserved sperm samples have been done immediately after thawing and compared with control and fresh semen. The extenders containing propolis exhibited higher percentage motility and motility duration than control group (P < 0.05). Especially the group IV (0.8 mg ml⁻¹ propolis) and the group V (1 mg ml⁻¹ propolis) showed significant positive effects on both post thaw motility and hatching ability. The propolis maintained the integrity of the spermatozoa during the cryopreservation process. Evaluating with its contents, it has been shown that propolis is an appropriate cryoprotective agent in fish semen.     

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.     

Studies on the mechanism of capacitation. II. Evidence for lipid transfer between plasma membrane of rat sperm and serum albumin during capacitation in vitro

1. Evidence has been provided for the transfer of phosphatidyl[14C]choline and [3H]cholesterol between bovine serum albumin and cauda epididymal rat spermatozoa in Krebs-Ringer bicarbonate medium, which can promote sperm capacitation. 2. An analysis of the lipid composition in both albumin and spermatozoa revealed that phospholipid levels decreased in the protein and increased by roughly comparable amounts in sperm cells during incubation in vitro. 3. Cholesterol (free + ester) increased in albumin and decreased in spermatozoa. Changes in the amount of esterified cholesterol were solely responsible for the increase associated with albumin, whereas whole sperm cell extracts showed a significant decline in free cholesterol. 4. The composition of albumin-bound fatty acids did not alter appreciably as a result of incubation with spermatozoa. 5. Rates of [14C]palmitic acid utilization by spermatozoa suggest that lipid synthesis accounted for less than 5% of the changes observed under the conditions of this study. 6. These results are interpreted as broadly supporting our previous proposal that lipid exchange between albumin and sperm cells is implicated in sperm capacitation in vitro. Specifically, the results are compatible with the idea that a decreased cholesterol/phospholipid ratio in the sperm plasma membrane facilitates this transformation.     

Fatty chain composition of phospholipids in sea urchin spermatozoa

1. An analysis was made of lipids extracted from the spermatozoa of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina. 2. Nearly all the lipids from both species consisted of phospholipids (about 80%) and cholesterol (about 14%). Triglyceride and cholesterol ester were present in trace amounts. 3. The fatty acid composition of each phospholipid was determined by gas-liquid chromatography. In both species, the fatty acid consisting of phosphatidylethanolamine was of the unsaturated type for the most part, while cardiolipin was comprised to a considerable degree of saturated fatty acids. In phosphatidylcholine and phosphatidylserine from H. pulcherrimus sperm, unsaturated fatty acid content was somewhat higher than that in phospholipids from A. crassispina sperm.     

Evidence for phosphorylation in the MSP cytoskeletal filaments of amoeboid spermatozoa

Nematode spermatozoa are highly specialized cells that lack flagella and, instead, extend a pseudopod to initiate motility. Crawling spermatozoa display classic features of amoeboid motility (e.g. protrusion of a pseudopod that attaches to the substrate and the assembly and disassembly of cytoskeletal filaments involved in cell traction and locomotion), however, cytoskeletal dynamics in these cells are powered exclusively by Major Sperm Protein (MSP) rather than actin and no other molecular motors have been identified. Thus, MSP-based motility is regarded as a simple locomotion machinery suitable for the study of plasma membrane protrusion and cell motility in general. This recent focus on MSP dynamics has increased the necessity of a standardized methodology to obtain C. elegans sperm extract that can be used in biochemical assays and proteomic analysis for comparative studies. In the present work we have modified a method to reproducibly obtain relative high amounts of proteins from C. elegans sperm extract. We show that these extracts share some of the properties observed in sperm extracts from the parasitic nematode Ascaris including Major Sperm Protein (MSP) precipitation and MSP fiber elongation. Using this method coupled to immunoblot detection, Mass Spectrometry identification, in silico prediction of functional domains and biochemical assays, our results indicate the presence of phosphorylation sites in MSP of Caenorhabditis elegans spermatozoa.     

The biflagellate spermatozoa of Colostethus marchesianus (Melin, 1941) (Anura, Dendrobatidae) from the type locality and of Colostethus sp. (aff. Marchesianus.) from a different locality: A scanning and transmission electron microscopy analysis

Scanning and transmission electron microscopy and fluorochrome staining with DAPI were used to study the spermatozoa of Colostethus marchesianus from the type locality and Colostethus sp. (aff. marchesianus) from a different locality. The sperm cell of these species resembles those of most neobatrachian species, except for the presence of two complete flagella, a character previously described only for four unrelated anuran species belonging to the families Leptodactylidae, Rhacophoridae and Dendrobatidae. Although very similar, the spermatozoa described here showed slight differences in the length of their nucleus, in the extent of the subacrosomal cone over the nucleus, and in the curvature of the axial fiber seen in cross section. The absence of a juxtaxonemal fiber and the presence of a comma shaped axial fiber seem to be common characteristics of dendrobatid spermatozoa. The minor differences found in our study may indicate that the taxa studied here are distinct species. The evolutionary significance of spermatozoa biflagellarity is still unclear, although the presence of this trait in unrelated groups suggests independent origins.     

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.