Contributions of extracellular and intracellular Ca2+ to regulation of sperm motility: Release of intracellular stores can hyperactivate CatSper1 and CatSper2 null sperm

In order to fertilize, mammalian sperm must hyperactivate. Hyperactivation is triggered by increased flagellar Ca(2+), which switches flagellar beating from a symmetrical to an asymmetrical pattern by increasing bending to one side. Thimerosal, which releases Ca(2+) from internal stores, induced hyperactivation in mouse sperm within seconds, even when extracellular Ca(2+) was buffered with BAPTA to approximately 30 nM. In sperm from CatSper1 or CatSper2 null mice, which lack functional flagellar alkaline-activated calcium currents, 50 microM thimerosal raised the flagellar bend amplitudes from abnormally low levels to normal pre-hyperactivated levels and, in 20-40% of sperm, induced hyperactivation. Addition of 1 mM Ni(2+) diminished the response. This suggests that intracellular Ca(2+) is abnormally low in the null sperm flagella. When intracellular Ca(2+) was reduced by BAPTA-AM in wild-type sperm, they exhibited flagellar beat patterns more closely resembling those of null sperm. Altogether, these results indicate that extracellular Ca(2+) is required to supplement store-released Ca(2+) to produce maximal and sustained hyperactivation and that CatSper1 and CatSper2 are key elements of the major Ca(2+) entry pathways that support not only hyperactivated motility but possibly also normal pre-hyperactivated motility.     

A novel function for CRISP1 in rodent fertilization: involvement in sperm-zona pellucida interaction

Epididymal protein CRISP1 participates in rat and mouse gamete fusion through its interaction with complementary sites on the egg surface. Based on in vivo observations, in the present study we investigated the possibility that CRISP1 plays an additional role in the sperm-zona pellucida (ZP) interaction that precedes gamete fusion. In vitro fertilization experiments using zona-intact rat and mouse eggs indicated that the presence of either an antibody against rat CRISP1 (anti-CRISP1) or rat native CRISP1 (rCRISP1) during gamete co-incubation produced a significant decrease in the percentage of fertilized eggs. However, differently to that expected for a protein involved in gamete fusion, no accumulation of perivitelline sperm was observed, suggesting that the inhibitions occurred at the sperm-ZP interaction level. Bacterially expressed recombinant CRISP1 (recCRISP1) also significantly inhibited egg fertilization. In this case, however, an increase in the number of perivitelline sperm was observed. Subsequent experiments evaluating the effect of anti-CRISP1 or rCRISP1 on the number of sperm bound per egg indicated that the protein is involved in the initial step of sperm-ZP binding. In agreement with these functional studies, indirect immunofluorescence experiments revealed that although rCRISP1 is capable of binding to both the ZP and the oolema, recCRISP1 only binds to the egg surface. The finding that deglycosylated rCRISP1 behaves as the untreated protein, whereas the heat-denatured rCRISP1 associated only with the oolema, indicates that the protein ZP-binding ability resides in the conformation rather than in the glycosydic portion of the molecule. The interaction between rCRISP1 and the ZP reproduces the sperm-ZP-binding behavior, as judged by the failure of the protein to interact with the ZP of fertilized eggs. Together, these results support the idea that CRISP1 participates not only in sperm-egg fusion but also in the prior stage of sperm-ZP interaction.     

Human sperm centrosomal function during fertilization, a novel assessment for male sterility

In human fertilization, the sperm introduces the centrosome-the microtubule organizing center-and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, the sperm aster, the functioning of which is essential for pronuclear movement for the union of the male and female genomes. We established functional assay for human sperm centrosomal function, by using heterologus ICSI system with bovine and rabbit eggs. After human sperm incorporation into mammalian egg, we observed that the sperm aster was organized from sperm centrosome, and the sperm aster enlarged as the sperm nuclei underwent pronuclear formation. The normal human sperm aster formation rate at 6 h post-ICSI were 60.0% in bovine egg and 36.1% in rabbit egg, respectively. However, sperm aster formation rate following heterologus ICSI into bovine eggs with teratozoospermia (globozoospermia, dysplasia of fibrous sheath) were low. These data indicate that human sperm centrosomal function is low in abnormal shaped sperm. Wherus, elucidation of human sperm centrosomal function can lead us to find a new type of failure in "post ICSI events in fertilization".     

Gle1 is a multifunctional DEAD-box protein regulator that modulates Ded1 in translation initiation

DEAD-box protein (Dbp) family members are essential for gene expression; however, their precise roles and regulation are not fully defined. During messenger (m)RNA export, Gle1 bound to inositol hexakisphosphate (IP(6)) acts via Dbp5 to facilitate remodeling of mRNA-protein complexes. In contrast, here we define a novel Gle1 role in translation initiation through regulation of a different DEAD-box protein, the initiation factor Ded1. We find that Gle1 physically and genetically interacts with Ded1. Surprisingly, whereas Gle1 stimulates Dbp5, it inhibits Ded1 ATPase activity in vitro, and IP(6) does not affect this inhibition. Functionally, a gle1-4 mutant specifically suppresses initiation defects in a ded1-120 mutant, and ded1 and gle1 mutants have complementary perturbations in AUG start site recognition. Consistent with this role in initiation, Gle1 inhibits translation in vitro in competent extracts. These results indicate that Gle1 has a direct role in initiation and negatively regulates Ded1. Together, the differential regulation of two distinct DEAD-box proteins by a common factor (Gle1) establishes a new paradigm for controlling gene expression and coupling translation with mRNA export.     

Identification of apolipoprotein A1 and immunoglobulin as components of a serum complex that mediates activation of human sperm motility

Serum is superior to other body fluids in activating the progressive motility of human spermatozoa and is used in connection with sperm separation for fertilization in vitro. The major activating capacity is localized to a macromolecular fraction, purified to homogeneity by a four-step protocol with ion-exchange chromatography, chromatofocusing, exclusion FPLC (elution corresponding to a molecular mass of about 250 kDa), and Blue Sepharose chromatography (no binding but elimination of albumin). The pure protein, at a concentration of 20-70 nmol/L, activated the motility to the same extent as serum. SDS-polyacrylamide gel electrophoresis under nonreducing conditions showed one band corresponding to a molecular mass of about 180 kDa. In the presence of mercaptoethanol, two bands are obtained corresponding to 50 kDa and about 25 kDa, respectively. Without the Blue Sepharose step, the sample after reduction revealed an additional band at about 67 kDa, suggesting that the fraction is then in complex also with albumin. Amino acid sequence analysis of the Blue Sepharose eluate identified three protein chains--those of apolipoprotein A1 and immunoglobulin heavy and light chains--suggesting that the preparation is an apolipoprotein A1-immunoglobulin complex. Antiserum raised toward the pure preparation in a rabbit inhibited human sperm motility, when added directly to spermatozoa. Pretreatment of human serum with rabbit antiserum significantly reduced its ability to activate sperm motility. The sperm activating capacity of the protein complex was destroyed by heating at 100 degrees C for 5 min, suggesting that the activity was dependent on intact protein conformations. Albumin, apolipoprotein A1, and immunoglobulins by themselves had only minor effects on sperm motility.(ABSTRACT TRUNCATED AT 250 WORDS)     

Egg and sperm recognition systems during fertilization

Fertilization is a programmed process that has many molecules and sequential events amenable to study. The biochemistry of fertilization has identified cellular and acellular components fundamental to the interactions between sperm and egg. Recent studies highlight the molecular details of the species-specificity of fertilization that involve protein–protein and protein–carbohydrate interactions. Although the diversity of structure and mechanism may imply rapid evolution of fertilization proteins, understanding the structure–function relationships has become important. Here, we introduce the molecules controlling the sperm AR, sperm attachment to, and penetration through, the egg investments.     

Sphingosine 1-phosphate as a novel immune regulator of dendritic cells

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to be involved in several physiological and pathological functions as an extracellular mediator. S1P receptors are widely expressed and thought to regulate important functions in cell signalling. Recently, the role of S1P on the immune system has evoked great interest. In particular, several aspects of the effects on antigen-presenting cells (APCs) as dendritic cells (DC) in mice and humans have been reported. In this review, we focus on the role played by S1P on the DC system and its effects in immune-related pathological states.     

Sphingosine 1-phosphate as a novel immune regulator of dendritic cells

Although originally described as an intracellular second messenger, sphingosine 1-phosphate (S1P) has recently been shown to be involved in several physiological and pathological functions as an extracellular mediator. S1P receptors are widely expressed and thought to regulate important functions in cell signalling. Recently, the role of S1P on the immune system has evoked great interest. In particular, several aspects of the effects on antigen-presenting cells (APCs) as dendritic cells (DC) in mice and humans have been reported. In this review, we focus on the role played by S1P on the DC system and its effects in immune-related pathological states.     

CyrA, a matricellular protein that modulates cell motility in Dictyostelium discoideum

CyrA, an extracellular matrix (slime sheath), calmodulin (CaM)-binding protein in Dictyostelium discoideum, possesses four tandem EGF-like repeats in its C-terminus and is proteolytically cleaved during asexual development. A previous study reported the expression and localization of CyrA cleavage products CyrA-C45 and CyrA-C40. In this study, an N-terminal antibody was produced that detected the full-length 63kDa protein (CyrA-C63). Western blot analyses showed that the intracellular expression of CyrA-C63 peaked between 12 and 16h of development, consistent with the time that cells are developing into a motile, multicellular slug. CyrA immunolocalization and CyrA-GFP showed that the protein localized to the endoplasmic reticulum, particularly its perinuclear component. CyrA-C63 secretion began shortly after the onset of starvation peaking between 8 and 16h of development. A pharmacological analysis showed that CyrA-C63 secretion was dependent on intracellular Ca(2+) release and active CaM, PI3K, and PLA2. CyrA-C63 bound to CaM both intra- and extracellularly and both proteins were detected in the slime sheath deposited by migrating slugs. In keeping with its purported function, CyrA-GFP over-expression enhanced cAMP-mediated chemotaxis and CyrA-C45 was detected in vinculin B (VinB)-GFP immunoprecipitates, thus providing a link between the increase in chemotaxis and a specific cytoskeletal component. Finally, DdEGFL1-FITC was detected on the membranes of cells capped with concanavalin A suggesting that a receptor exists for this peptide sequence. Together with previous studies, the data presented here suggests that CyrA is a bona fide matricellular protein in D. discoideum.     

CCN3 modulates bone turnover and is a novel regulator of skeletal metastasis

The CCN family of proteins is composed of six secreted proteins (CCN1-6), which are grouped together based on their structural similarity. These matricellular proteins are involved in a large spectrum of biological processes, ranging from development to disease. In this review, we focus on CCN3, a founding member of this family, and its role in regulating cells within the bone microenvironment. CCN3 impairs normal osteoblast differentiation through multiple mechanisms, which include the neutralization of pro-osteoblastogenic stimuli such as BMP and Wnt family signals or the activation of pathways that suppress osteoblastogenesis, such as Notch. In contrast, CCN3 is known to promote chondrocyte differentiation. Given these functions, it is not surprising that CCN3 has been implicated in the progression of primary bone cancers such as osteosarcoma, Ewing’s sarcoma and chondrosarcoma. More recently, emerging evidence suggests that CCN3 may also influence the ability of metastatic cancers to colonize and grow in bone.     

Toxoplasma as a novel system for motility

Motility is a characteristic of most living organisms and often requires specialized structures like cilia or flagella. An alternative is amoeboid movement, where the polymerization/depolymerization of actin leads to the formation of pseudopodia, filopodia and/or lamellipodia that enable the cell to crawl along a surface. Despite their lack of locomotive organelles and in absence of cell deformation, members of the apicomplexan parasites employ a unique form of locomotion called gliding motility to promote their migration across biological barriers and to power host-cell invasion and egress. Detailed studies in Toxoplasma gondii and Plasmodium species have revealed that this unique mode of movement is dependent on a myosin of class XIV and necessitates actin dynamics and the concerted discharge and processing of adhesive proteins. Gliding is essential for the survival and infectivity of these obligate intracellular parasites, which cause severe disease in humans and animals.     

Evidence for the involvement of zinc in the association of CRISP1 with rat sperm during epididymal maturation

Rat epididymal protein CRISP1 (cysteine-rich secretory protein 1) associates with sperm during maturation and participates in fertilization. Evidence indicates the existence of two populations of CRISP1 in sperm: one loosely bound and released during capacitation, and one strongly bound that remains after this process. However, the mechanisms underlying CRISP1 binding to sperm remain mostly unknown. Considering the high concentrations of Zn(2+) present in the epididymis, we investigated the potential involvement of this cation in the association of CRISP1 with sperm. Caput sperm were coincubated with epididymal fluid in the presence or absence of Zn(2+), and binding of CRISP1 to sperm was examined by Western blot analysis. An increase in CRISP1 was detected in sperm exposed to Zn(2+), but not if the cation was added with ethylenediaminetetra-acetic acid (EDTA). The same results were obtained using purified CRISP1. Association of CRISP1 with sperm was dependent on epididymal fluid and Zn(2+) concentrations and incubation time. Treatment with NaCl (0.6 M) removed the in vitro-bound CRISP1, indicating that it corresponds to the loosely bound population. Flow cytometry of caput sperm exposed to biotinylated CRISP1/avidin-fluorescein isothiocyanate revealed that only the cells incubated with Zn(2+) exhibited an increase in fluorescence. When these sperm were examined by epifluorescence microscopy, a clear staining in the tail, accompanied by a weaker labeling in the head, was observed. Detection of changes in the tryptophan fluorescence emission spectra of CRISP1 when exposed to Zn(2+) supported a direct interaction between CRISP1 and Zn(2+). Incubation of either cauda epididymal fluid or purified CRISP1 with Zn(2+), followed by native-PAGE and Western blot analysis, revealed the presence of high-molecular-weight CRISP1 complexes not detected in fluids treated with EDTA. Altogether, these results support the involvement of CRISP1-Zn(2+) complexes in the association of the loosely bound population of CRISP1 with sperm during epididymal maturation.     

Phosphotyrosine signalling as a regulator of neural crest cell adhesion and motility

We demonstrate that neural crest cell-cell adhesion, cell-substrate adhesion, and ultimately cell motility, are highly dependent on the balanced action of tyrosine kinases and tyrosine phosphatases. Neural crest cell migration on fibronectin is diminished in the presence of the tyrosine phosphatase inhibitor vanadate or tyrosine kinase inhibitor herbimycin A, while cadherin-rich cell-cell adhesions are significantly increased. In contrast, cells treated with the kinase inhibitor genistein have decreased motility, rearrange rapidly and reversibly into a pavement-like monolayer, but have no increase in cadherin interactions. Genistein-sensitive tyrosine kinases may therefore abrogate a latent sensitivity of neural crest cells to contact-mediated inhibition of movement. Furthermore, we show that the activity of herbimycin A-sensitive kinases is necessary for focal adhesion formation in these cells. Moreover, the size and distribution of these adhesions are acutely sensitive to the actions of tyrosine phosphatases and genistein-sensitive kinases. We propose that in migrating neural crest cells there is a balance in phosphotyrosine signalling which minimises both cell-cell adhesion and contact inhibition of movement, while enhancing dynamic cell-substrate interactions and thus the conditions for motility.     

Identification of a novel sperm class and its role in fertilization in Drosophila

In many species, males have evolved to produce a sterile sperm (parasperm) in conjunction with fertilizing sperm (eusperm). Here, we document evidence of males depositing two morphologically distinct types of parasperm (1 and 2) into the female reproductive tract in Drosophila pseudoobscura. These parasperm differ in length, shape, amount produced, amount in long‐term storage and may have separate roles in ensuring male fertilization success. Although both parasperm types protect eusperm from female spermicides, only parasperm 2, which has a corkscrew shape, is associated with sperm competition. Increased production of parasperm 2 is also negatively correlated with the eusperm and parasperm 1 production. Thus, selection may be acting on parasperm production in the presence of sperm competition. Our findings show how both sperm competition and cryptic female choice may be acting in conjunction to influence the evolution of ejaculate composition. Our identification and characterization of two distinct parasperm morphs will enhance the ability for further evaluation of parasperm's role in fertilization.