Role of sperm surface arylsulfatase A in mouse sperm-zona pellucida binding

We have previously described the zonae pellucidae (ZP) binding ability of a pig sperm surface protein, P68. Our recent results on peptide sequencing of 3 P68 tryptic peptides and molecular cloning of pig testis arylsulfatase A (AS-A) revealed the identity of P68 as AS-A. In this report, we demonstrate the presence of AS-A on the mouse sperm surface and its role in ZP binding. Using anti-AS-A antibody, we have shown by immunoblotting that AS-A was present in a Triton X-100 extract of mouse sperm. The presence of AS-A on the sperm plasma membrane was conclusively demonstrated by indirect immunofluorescence, immunogold electron microscopy, and AS-A's desulfation activity on live mouse sperm. The AS-A remained on the head surface of in vivo capacitated sperm, as revealed by positive immunofluorescent staining of oviductal/uterine sperm. Significantly, the role of mouse sperm surface AS-A on ZP binding was demonstrated by dose-dependent decreases of sperm-ZP binding on sperm pretreatment with anti-AS-A IgG/Fab. Furthermore, Alexa-430 conjugated AS-A bound to mouse ZP of unfertilized eggs but not to fertilized ones, and this level of binding increased and approached saturation with increasing Alexa-430 AS-A concentrations. Moreover, in vivo fertilization was markedly decreased when mouse sperm pretreated with anti-AS-A IgG were artificially inseminated into females. All of these results designated a new function for AS-A in mouse gamete interaction.     

Spermatogenesis and distinctive mature sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

The structures of differentiating male germ cells in the testis of the giant freshwater prawn, Macrobrachium rosenbergii, were studied by light and electron microscopy. Based on ultrastructural characteristics, the developing male germ cells are classified into 12 stages, including spermatogonia, six phases of primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase), secondary spermatocyte, three stages of spermatids and mature sperm. During spermatogenesis, the differentiating germ cells have characteristics similar to those of other invertebrates, but they exhibit some unique characteristics during spermiogenesis. In particular, an early spermatid has a round nucleus with highly condensed heterochromatin, appearing as thick interconnecting cords throughout the nucleus. In contrast to most invertebrates and vertebrates, the chromatin begins to decondense in one-half of the nucleus at the mid spermatid stage. In the late spermatid, the chromatin becomes almost entirely decondensed with only a small crescent-shaped heterochromatin patch remaining at the anterior pole of the nucleus. Mature sperm possess an everted umbrella-shaped plate with a spike covering the anterior pole of the nucleus, whose chromatin is totally decondensed as only small traces of histones H3 and H2B remain. The acrosome appears at the ruffled border of the spike plate as small sac-like structures. Few mitochondria remain in the cytoplasm at the posterior pole.     

Structural Evidence of Glycoprotein Assembly in Cellular Membrane Compartments prior to Alphavirus Budding

Membrane glycoproteins of alphavirus play a critical role in the assembly and budding of progeny virions. However, knowledge regarding transport of viral glycoproteins to the plasma membrane is obscure. In this study, we investigated the role of cytopathic vacuole type II (CPV-II) through in situ electron tomography of alphavirus-infected cells. The results revealed that CPV-II contains viral glycoproteins arranged in helical tubular arrays resembling the basic organization of glycoprotein trimers on the envelope of the mature virions. The location of CPV-II adjacent to the site of viral budding suggests a model for the transport of structural components to the site of budding. Thus, the structural characteristics of CPV-II can be used in evaluating the design of a packaging cell line for replicon production.Semliki Forest virus (SFV) is an enveloped alphavirus belonging to the family Togaviridae. This T=4 icosahedral virus particle is approximately 70 nm in diameter (30) and consists of 240 copies of E1/E2 glycoprotein dimers (3, 8, 24). The glycoproteins are anchored in a host-derived lipid envelope that encloses a nucleocapsid, made of a matching number of capsid proteins and a positive single-stranded RNA molecule. After entry of the virus via receptor-mediated endocytosis, a low-pH-induced fusion of the viral envelope with the endosomal membrane delivers the nucleocapsid into the cytoplasm, where the replication events of SFV occur (8, 19, 30). Replication of the viral genome and subsequent translation into structural and nonstructural proteins followed by assembly of the structural proteins and genome (7) lead to budding of progeny virions at the plasma membrane (18, 20). The synthesis of viral proteins shuts off host cell macromolecule synthesis, which allows for efficient intracellular replication of progeny virus (7). The expression of viral proteins leads to the formation of cytopathic vacuolar compartments as the result of the reorganization of cellular membrane in the cytoplasm of an infected cell (1, 7, 14).Early studies using electron microscopy (EM) have characterized the cytopathic vacuoles (CPVs) in SFV-infected cells (6, 13, 14) and identified two types of CPV, namely, CPV type I (CPV-I) and CPV-II. It was found that CPV-I is derived from modified endosomes and lysosomes (18), while CPV-II is derived from the trans-Golgi network (TGN) (10, 11). Significantly, the TGN and CPV-II vesicles are the major membrane compartments marked with E1/E2 glycoproteins (9, 11, 12). Inhibition by monensin results in the accumulation of E1/E2 glycoproteins in the TGN (12, 26), thereby indicating the origin of CPV-II. While CPV-II is identified as the predominant vacuolar structure at the late stage of SFV infection, the exact function of this particular cytopathic vacuole is less well characterized than that of CPV-I (2, 18), although previous observations have pointed to the involvement of CPV-II in budding, because an associated loss of viral budding was observed when CPV-II was absent (9, 36).In this study, we characterized the structure and composition of CPV-II in SFV-infected cells in situ with the aid of electron tomography and immuno-electron microscopy after physical fixation of SFV-infected cells by high-pressure freezing and freeze substitution (21, 22, 33). The results revealed a helical array of E1/E2 glycoproteins within CPV-II and indicate that CPV-II plays an important role in intracellular transport of glycoproteins prior to SFV budding.     

Comparative analysis of the Spirulina platensis subcellular proteome in response to low- and high-temperature stresses: uncovering cross-talk of signaling components

The present study focused on comparative proteome analyses of low- and high-temperature stresses and potential protein-protein interaction networks, constructed by using a bioinformatics approach, in response to both stress conditions. The data revealed two important points: first, the results indicate that low-temperature stress is tightly linked with oxidative stress as well as photosynthesis; however, no specific mechanism is revealed in the case of the high-temperature stress response. Second, temperature stress was revealed to be linked with nitrogen and ammonia assimilation. Moreover, the data also highlighted the cross-talk of signaling pathways. Some of the detected signaling proteins, e.g., Hik14, Hik26 and Hik28, have potential interactions with differentially expressed proteins identified in both temperature stress conditions. Some differentially expressed proteins found in the Spirulina protein-protein interaction network were also examined for their physical interactions by a yeast two hybrid system (Y2H). The Y2H results obtained in this study suggests that the potential PPI network gives quite reliable potential interactions for Spirulina. Therefore, the bioinformatics approach employed in this study helps in the analysis of phenomena where proteome analyses of knockout mutants have not been carried out to directly examine for specificity or cross-talk of signaling components.     

Detection of nonribosomal peptide synthetase genes in Xylaria sp. BCC1067 and cloning of XyNRPSA

Nonribosomal peptides, synthesized by nonribosomal peptide synthetases (NRPS), are an important group of diverse bioactive fungal metabolites. Xylaria sp. BCC1067, which is known to produce a variety of biologically active metabolites, was studied for gene encoding NRPS by two different PCR-based methods and seven different NRPS fragments were obtained. In addition, screening a genomic library with an amplified NRPS fragment as a probe identified a putative NRPS gene named XyNRPSA. The functionality of XyNRPSA for the production of a corresponding metabolite was probed by gene insertion inactivation. Comparing the disrupting metabolite profile with that of the wild type led to the identification of a speculated metabolite. The crude extract of Xylaria sp. BCC1067 also exhibits antifungal activity against the human pathogens Candida albicans and Trichophyton mentagrophytes. However, the evaluation of biological activity of the XyNRPSA product suggests that it is neither a compound with antifungal activity nor a siderophore. In the vicinity of XyNRPSA, a second gene (named XyPtB) was identified. Its localization and homology to orfB of the ergot alkaloid biosynthetic gene cluster suggests that XyPtB may be involved in XyNRPSA product biosynthesis.     

Sperm from mice genetically deficient for the PCSK4 proteinase exhibit accelerated capacitation, precocious acrosome reaction, reduced binding to egg zona pellucida, and impaired fertilizing ability

The gene for proprotein convertase subtilisin/kexin-like 4 (PCSK4, previously known as PC4) is primarily transcribed in testicular spermatogenic cells. Its inactivation in mouse causes severe male subfertility. To better understand the reproductive function of PCSK4, we examined its subcellular localization in the testicular epithelium via immunohistochemistry, and on intact sperm via indirect immunofluorescence and immunoelectron microscopy. PCSK4 was detected in the acrosomal granules of round spermatids, in the acrosomal ridges of elongated spermatids, and on the sperm plasma membrane overlying the acrosome. We also investigated PCSK4 relevance for sperm acquisition of fertilizing ability by comparing wild-type and PCSK4-null sperm for their abilities in capacitation, acrosome reaction, and egg binding in vitro. PCSK4-null sperm underwent capacitation at a faster rate; they were induced to acrosome react by lower concentrations of zona pellucida; and their egg-binding ability was only half that of wild-type sperm. These sperm physiologic anomalies likely contribute to the severe subfertility of PCSK4-deficient male mice.     

Sperm capacitation induces an increase in lipid rafts having zona pellucida binding ability and containing sulfogalactosylglycerolipid

Sperm gain full ability to bind to the zona(e) pellucida(e) (ZP) during capacitation. Since lipid rafts are implicated in cell adhesion, we determined whether capacitated sperm lipid rafts had affinity for the ZP. We demonstrated that lipid rafts, isolated as low-density detergent resistant membranes (DRMs), from capacitated pig sperm had ability to bind to homologous ZP. This binding was dependent on pig ZPB glycoprotein, a major participant in sperm binding. Capacitated sperm DRMs were also enriched in the male germ cell specific sulfogalactosylglycerolipid (SGG), which contributed to DRMs-ZP binding. Furthermore, SGG may participate in the formation of sperm DRMs due to its interaction with cholesterol, an integral component of lipid rafts, as shown by infrared spectroscopic studies. Since sperm capacitation is associated with cholesterol efflux from the sperm membrane, we questioned whether the formation of DRMs was compromised in capacitated sperm. Our studies indeed revealed that capacitation induced increased levels of sperm DRMs, with an enhanced ZP affinity. These results corroborated the implication of lipid rafts and SGG in cell adhesion and strongly suggested that the enhanced ZP binding ability of capacitated sperm may be attributed to increased levels and a greater ZP affinity of lipid rafts in the sperm plasma membrane.     

Effect of progesterone administration on the distribution of oviductal carbohydrates in <Emphasis Type="Italic">Rana tigrina</Emphasis>

Our aim has been to determine whether carbohydrate distribution in the oviducts of progesterone-treated animals is comparable with that of seasonal breeders in Rana tigrina. Like many other anurans, R. tigrina oviduct exhibits a short straight portion (pars recta, pr) at the beginning followed by a long, highly coiled portion (pars convoluta, pc). Histologically, the oviduct of this species revealed some unique features, one of which was intense toluidine blue staining, specifically in the upper mucosal glands of pc4. Based on lectin reactivities in the epithelial cells and mucosal glands, patterns of lectin staining in the seasonal breeders were classified into seven types: R1-R3 (for pr) and C1-C4 (for pc). Typically, some lectins reacted selectively either with ciliated cells (concanavalin A) or non-cialiated cells (Ricinus communis agglutinin I and wheatgerm agglutinin); however, Bandeiraea simplicifolia agglutinin I reacted with both cell types. These staining patterns were different in the progesterone-treated animals. Differences in glycan distribution in the oviductal secretions were revealed by lectin blotting. Compared with the seasonal breeders, an enhanced staining of some lectins was noted in the hormone-treated animals: either an increased staining intensity of existing protein bands or additional staining of new protein bands. Inversely, the staining of wheatgerm agglutinin was markedly diminished in the hormone-treated animals, suggesting the inhibitory effect of progesterone on oviductal glycan distribution. Whether alteration in glycan distribution upon progesterone treatment affects the physiological properties of the released jelly substances remains to be addressed. This research was supported by Thailand Research Funds (to W.W.), a Research Initiate Grant from Kasetsart University (to A.T.), and Mahidol University.     

Percoll gradient-centrifuged capacitated mouse sperm have increased fertilizing ability and higher contents of sulfogalactosylglycerolipid and docosahexaenoic acid-containing phosphatidylcholine compared to washed capacitated mouse sperm

Although Percoll gradient centrifugation has been used routinely to prepare motile human sperm, its use in preparing motile mouse sperm has been limited. Here, we showed that Percoll gradient-centrifuged (PGC) capacitated mouse sperm had markedly higher fertilizing ability (sperm-zona pellucida [ZP] binding and in vitro fertilization) than washed capacitated mouse sperm. We also showed that the lipid profiles of PGC capacitated sperm and washed capacitated sperm differed significantly. The PGC sperm had much lower contents of cholesterol and phospholipids. This resulted in relative enrichment of male germ cell-specific sulfogalactosylglycerolipid (SGG), a ZP-binding ligand, in PGC capacitated sperm, and this would explain, in part, their increased ZP-binding ability compared with that of washed capacitated sperm. Analyses of phospholipid fatty acyl chains revealed that PGC capacitated sperm were enriched in phosphatidylcholine (PC) molecular species containing highly unsaturated fatty acids (HUFAs), with docosahexaenoic acid (DHA; C22: 6n-3) being the predominant HUFA (42% of total hydrocarbon chains of PC). In contrast, the level of PC-HUFAs comprising arachidonic acid (20:4n-6), docosapentaenoic acid (C22:5n-6), and DHA in washed capacitated sperm was only 27%. Having the highest unsaturation degree among all HUFAs in PC, DHA would enhance membrane fluidity to the uppermost. Therefore, membranes of PGC capacitated sperm would undergo fertilization-related fusion events at higher rates than washed capacitated sperm. These results suggested that PGC mouse sperm should be used in fertilization experiments and that SGG and DHA should be considered to be important biomarkers for sperm fertilizing ability.