Arachidonic acid-induced acrosomal loss in the spermatozoa of a marsupial, the tammar wallaby (Macropus eugenii)

1997 ◽  
Vol 9 (8) ◽  
pp. 803 ◽  
Author(s):  
Yulia Sistina ◽  
John C. Rodger
Keyword(s):  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Tammar Wallaby ◽  
Multiple Point ◽  
Fusion Event ◽  
Macropus Eugenii ◽  
Kinase C ◽  
Acrosomal Membrane ◽  
Late Stages

Tammar wallaby spermatozoa were induced to undergo acrosomal loss when incubated with arachidonic acid (AA). Ultrastructural examination indicated that the AA-induced acrosomal loss occurred via multiple point fusions between the outer acrosomal membrane and the overlying plasma membrane. This form of acrosomal loss mimicked the physiological acrosome reaction (AR) seen in the sperm of eutherian mammals. The fusion event was limited to the acrosomal region of the plasma membrane and did not proceed past the peri-acrosomal ring. The entire acrosome was lost after AA treatment leaving no evidence of a persistent equatorial segment-like region. Ultrastructural evidence of AR-like membrane fusion was seen immediately on addition of 50 µg mL-1 AA and a large proportion of sperm examined after five min incubation were in the late stages of membrane fusion. Longer-term incubation with AA had deleterious effects on wallaby sperm motility. It remains to be determined whether the AA-induced membrane fusion observed here indicates that AA is involved in the marsupial AR. However, pretreatment of sperm with the protein kinase C (PKC) inhibitor HMG significantly reduced AA-induced acrosomal loss suggesting that AA may have acted via PKC. If this is so, AA is probably physiologically significant and a novel pathway may be operating during AR induction in marsupials.

scholarly journals Autoradiographic visualization of the mouse egg's sperm receptor bound to sperm.

1986 ◽  
Vol 102 (4) ◽  
pp. 1363-1371 ◽  
Author(s):  
J D Bleil ◽  
P M Wassarman
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Present Report ◽  
Fusion Event ◽  
Acrosomal Membrane ◽  
Specific Receptors ◽  
Species Specific ◽  
Mammalian Eggs ◽  
Silver Grains ◽  
Sperm Receptor

The extracellular coat, or zona pellucida, of mammalian eggs contains species-specific receptors to which sperm bind as a prelude to fertilization. In mice, ZP3, one of only three zona pellucida glycoproteins, serves as sperm receptor. Acrosome-intact, but not acrosome-reacted, mouse sperm recognize and interact with specific O-linked oligosaccharides of ZP3 resulting in sperm-egg binding. Binding, in turn, causes sperm to undergo the acrosome reaction; a membrane fusion event that results in loss of plasma membrane at the anterior region of the head and exposure of inner acrosomal membrane with its associated acrosomal contents. Bound, acrosome-reacted sperm are able to penetrate the zona pellucida and fuse with the egg's plasma membrane (fertilization). In the present report, we examined binding of radioiodinated, purified, egg ZP3 to both acrosome intact and acrosome reacted sperm by whole-mount autoradiography. Silver grains due to bound 125I-ZP3 were found localized to the acrosomal cap region of heads of acrosome-reacted sperm. Under the same conditions, 125I-fetuin bound at only bacKground levels to heads of both acrosome-intact and -reacted sperm, and 125I-ZP2, another zona pellucida glycoprotein, bound preferentially to acrosome-reacted sperm. These results provide visual evidence that ZP3 binds preferentially and specifically to heads of acrosome intact sperm; properties expected of the mouse egg's sperm receptor.

Tracing sperm acrosome differentiation in the testis and maturation in the epididymis of the tammar wallaby (Macropus eugenii) with a 45-kDa acrosome-membrane-associated protein

2002 ◽  
Vol 14 (2) ◽  
pp. 69
Author(s):  
Xiyi Zhang ◽  
Minjie Lin
Keyword(s):  
Molecular Marker ◽  
Tammar Wallaby ◽  
Sperm Nucleus ◽  
Macropus Eugenii ◽  
Acrosomal Membrane ◽  
Epididymal Maturation ◽  
Associated Proteins ◽  
Inner Acrosomal Membrane ◽  
Sperm Acrosome

A 45-kDa protein was originally extracted from a depression, where the acrosome is lodged, on the anterior end of the sperm nucleus of ejaculated wallaby spermatozoa. Using immunofluorescent and confocal microscopes, this study demonstrates that the 45-kDa protein is persistently localized to the sperm acrosome throughout the periods of spermiogenesis, spermiation, epididymal maturation and ejaculation in the tammar wallaby. The distribution of the 45-kDa protein is always on the perimeter of the acrosome and associated with the acrosomal membrane, so that changes in the shape of the 45-kDa immunofluorescent labelling mirror changes in the shape of the acrosome during its differentiation in the testis and epididymis. Thus, the 45-kDa protein may be used as a molecular marker to study the marsupial acrosome differentiation and to chart the events of testicular and epididymal maturation of the spermatozoa. Furthermore, the behaviour of the 45-kDa protein during the immunostaining process suggests that this protein is a largely insoluble and detergent-resistant protein and may play an important role in attachment of the acrosome to the nucleus during sperm formation, similar to those inner acrosomal-membrane-associated proteins that have been reported in eutherian spermatozoa.

scholarly journals Prm1 Targeting to Contact Sites Enhances Fusion during Mating in Saccharomyces cerevisiae

2010 ◽  
Vol 9 (10) ◽  
pp. 1538-1548 ◽  
Author(s):  
Valerie N. Olmo ◽  
Eric Grote
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Intracellular Transport ◽  
Plasma Membranes ◽  
Yeast Cells ◽  
Fusion Event ◽  
Mating Activity ◽  
Content Type ◽  
Contact Sites

ABSTRACT Prm1 is a pheromone-regulated membrane glycoprotein involved in the plasma membrane fusion event of Saccharomyces cerevisiae mating. Although this function suggests that Prm1 should act at contact sites in pairs of mating yeast cells where plasma membrane fusion occurs, only a small percentage of the total Prm1 was actually detected on the plasma membrane. We therefore investigated the intracellular transport of Prm1 and how this transport contributes to cell fusion. Two Prm1 chimeras that were sorted away from the contact site had reduced fusion activity, indicating that Prm1 indeed functions at contact sites. However, most Prm1 is located in endosomes and other cytoplasmic organelles and is targeted to vacuoles for degradation. Mutations in a putative endocytosis signal in a cytoplasmic loop partially stabilized the Prm1 protein and caused it to accumulate on the plasma membrane, but this endocytosis mutant actually had reduced mating activity. When Prm1 was expressed from a galactose-regulated promoter and its synthesis was repressed at the start of mating, vanishingly small amounts of Prm1 protein remained at the time when the plasma membranes came into contact. Nevertheless, this stable pool of Prm1 was retained at polarized sites on the plasma membrane and was sufficient to promote plasma membrane fusion. Thus, the amount of Prm1 expressed in mating yeast is far in excess of the amount required to facilitate fusion.

scholarly journals Distinct Effects of Fatty Acids on Translocation of γ- and ε-Subspecies of Protein Kinase C

1998 ◽  
Vol 143 (2) ◽  
pp. 511-521 ◽  
Author(s):  
Yasuhito Shirai ◽  
Kaori Kashiwagi ◽  
Keiko Yagi ◽  
Norio Sakai ◽  
Naoaki Saito
Keyword(s):  
Fatty Acids ◽  
Arachidonic Acid ◽  
Plasma Membrane ◽  
Protein Kinase C ◽  
Linoleic Acid ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Target Site ◽  
Simultaneous Application ◽  
Kinase C

Effects of fatty acids on translocation of the γ- and ε-subspecies of protein kinase C (PKC) in living cells were investigated using their proteins fused with green fluorescent protein (GFP). γ-PKC–GFP and ε-PKC–GFP predominated in the cytoplasm, but only a small amount of γ-PKC–GFP was found in the nucleus. Except at a high concentration of linoleic acid, all the fatty acids examined induced the translocation of γ-PKC–GFP from the cytoplasm to the plasma membrane within 30 s with a return to the cytoplasm in 3 min, but they had no effect on γ-PKC–GFP in the nucleus. Arachidonic and linoleic acids induced slow translocation of ε-PKC–GFP from the cytoplasm to the perinuclear region, whereas the other fatty acids (except for palmitic acid) induced rapid translocation to the plasma membrane. The target site of the slower translocation of ε-PKC–GFP by arachidonic acid was identified as the Golgi network. The critical concentration of fatty acid that induced translocation varied among the 11 fatty acids tested. In general, a higher concentration was required to induce the translocation of ε-PKC–GFP than that of γ-PKC–GFP, the exceptions being tridecanoic acid, linoleic acid, and arachidonic acid. Furthermore, arachidonic acid and the diacylglycerol analogue (DiC8) had synergistic effects on the translocation of γ-PKC–GFP. Simultaneous application of arachidonic acid (25 μM) and DiC8 (10 μM) elicited a slow, irreversible translocation of γ-PKC– GFP from the cytoplasm to the plasma membrane after rapid, reversible translocation, but a single application of arachidonic acid or DiC8 at the same concentration induced no translocation. These findings confirm the involvement of fatty acids in the translocation of γ- and ε-PKC, and they also indicate that each subspecies has a specific targeting mechanism that depends on the extracellular signals and that a combination of intracellular activators alters the target site of PKCs.

scholarly journals Acrosome formation during sperm transit through the epididymis in two marsupials, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula)

1999 ◽  
Vol 194 (2) ◽  
pp. 223-232
Author(s):  
MINJIE LIN ◽  
JOHN C. RODGER
Keyword(s):  
Spatial Information ◽  
Dorsal Surface ◽  
Tammar Wallaby ◽  
Sperm Head ◽  
The Body ◽  
Brushtail Possum ◽  
Trichosurus Vulpecula ◽  
Macropus Eugenii ◽  
Acrosomal Membrane ◽  
Acrosome Formation

In certain Australian marsupials including the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula), formation of the acrosome is not completed in the testis but during a complex differentiation process as spermatozoa pass through the epididymis. Using transmission and scanning electron microscopy this paper defined the process of acrosome formation in the epididymis, providing temporal and spatial information on the striking reorganisation of the acrosomal membranes and matrix and of the overlying sperm surface involved. On leaving the testis wallaby and possum spermatozoa had elongated ‘scoop’-shaped acrosomes projecting from the dorsal surface of the head. During passage down the epididymis, this structure condensed into the compact button-like organelle found on ejaculated spermatozoa. This condensation was achieved by a complex process of infolding and fusion of the lateral projections of the ‘scoop’. In the head of the epididymis the rims of the lateral scoop projections became shorter and thickened and folded inwards, to eventually meet midway along the longitudinal axis of the acrosome. As spermatozoa passed through the body of the epididymis the lateral projections fused together. Evidence of this fusion of the immature outer acrosomal membrane is the presence of vesicles within the acrosomal matrix which persist even in ejaculated spermatozoa. When spermatozoa have reached the tail of the epididymis the acrosome condenses into its mature form, as a small button-like structure contained within the depression on the anterior end of the nucleus. During the infolding process, the membranes associated with the immature acrosome are either engulfed into the acrosomal matrix (outer acrosomal membrane), or eliminated from the sperm head as tubular membrane elements (cytoplasmic membrane). Thus the surface and organelles of the testicular sperm head are transient structures in those marsupials with posttesticular acrosome formation and this must be taken into consideration in attempts to dissect the cell and molecular biology of fertilisation.

scholarly journals Differential Regulation of Granule-to-Granule and Granule-to-Plasma Membrane Fusion during Secretion from Rat Pituitary Lactotrophs

2000 ◽  
Vol 150 (4) ◽  
pp. 839-848 ◽  
Author(s):  
Amanda J. Cochilla ◽  
Joseph K. Angleson ◽  
William J. Betz
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Calcium Ionophore ◽  
Differential Regulation ◽  
Cellular Mechanism ◽  
Extracellular Potassium ◽  
Kinase C ◽  
Compound Exocytosis ◽  
Rat Pituitary

We used fluorescence imaging of individual exocytic events together with electron microscopy to study the regulation of dense core granule-to-plasma membrane fusion and granule-to-granule fusion events that occur during secretion from rat pituitary lactotrophs. Stimulating secretion with elevated extracellular potassium, with the calcium ionophore ionomycin, or with thyrotropin releasing hormone or vasoactive intestinal polypeptide resulted in abundant exocytic structures. Approximately 67% of these structures consisted of multiple granules fused together sharing a single exocytic opening with the plasma membrane, i.e., compound exocytosis. For all of these stimulation conditions there appeared to be a finite number of plasma membrane fusion sites, ∼11 sites around each cellular equator. However, a granule could fuse directly with another granule that had already fused with the plasma membrane even before all plasma membrane sites were occupied. Granule-to-plasma membrane and granule-to-granule fusion events were subject to different regulations. Forskolin, which can elevate cAMP, increased the number of granule-to-granule fusion events without altering the number of granule-to-plasma membrane fusion events. In contrast, the phorbol ester PMA, which activates protein kinase C increased both granule-to-granule and granule-to-plasma membrane fusion events. These results provide a cellular mechanism that can account for the previously demonstrated potentiation of secretion from lactotrophs by cAMP- and PKC-dependent pathways.

scholarly journals Prm1 Prevents Contact-Dependent Lysis of Yeast Mating Pairs

2004 ◽  
Vol 3 (6) ◽  
pp. 1664-1673 ◽  
Author(s):  
Hui Jin ◽  
Candice Carlile ◽  
Scott Nolan ◽  
Eric Grote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fusion ◽  
Yeast Cells ◽  
Osmotic Gradient ◽  
Fusion Event ◽  
Mating Partner ◽  
Yeast Mating ◽  
Cell Wall Remodeling ◽  
A Cell

ABSTRACT Membrane fusion requires localized destabilization of two phospholipid bilayers, but unrestrained membrane destabilization could result in lysis. prm1 mutant yeast cells have a defect at the plasma membrane fusion stage of mating that typically results in the accumulation of prezygotes that have fingers of membrane-bound cytoplasm projecting from one cell of each pair into its mating partner in the direction of the osmotic gradient between the cells. However, some prm1 mating pairs fuse successfully whereas the two cells in other prm1 mating pairs simultaneously lyse. Lysis only occurs if both mating partners are prm1 mutants. Osmotic stabilization does not protect prm1 mating pairs from lysis, indicating that lysis is not caused by a cell wall defect. prm1 mating pairs without functional mitochondria still lyse, ruling out programmed cell death. No excess lysis was found after pheromone treatment of haploid prm1 cells, and lysis did not occur in mating pairs when prm1 was combined with the fus1 and fus2 mutations to block cell wall remodeling. Furthermore, short (<1 μm) cytoplasmic microfingers indicating the completion of cell wall remodeling appeared immediately before lysis. In combination, these results demonstrate that plasma membrane contact is a prerequisite for lysis. Cytoplasmic microfingers are unlikely to cause lysis since most prm1 mating pairs with microfingers do not lyse, and microfingers were also detected before fusion in some wild-type mating pairs. The lysis of prm1 mutant mating pairs suggests that the Prm1 protein stabilizes the membrane fusion event of yeast mating.

scholarly journals A specific interaction in vitro between pancreatic zymogen granules and plasma membranes: stimulation by G-protein activators but not by Ca2+.

1989 ◽  
Vol 109 (6) ◽  
pp. 2801-2808 ◽  
Author(s):  
C Y Nadin ◽  
J Rogers ◽  
S Tomlinson ◽  
J M Edwardson
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Specific Interaction ◽  
Zymogen Granules ◽  
Fusion Event ◽  
A Cell

The molecular details of the final step in the process of regulated exocytosis, the fusion of the membrane of the secretory granule with the plasma membrane, are at present obscure. As a first step in an investigation of this membrane fusion event, we have developed a cell-free assay for the interaction between pancreatic zymogen granules and plasma membranes. We show here that plasma membranes are able to trigger the release of the granule contents, and that this effect is specific to pancreatic membranes, involves membrane fusion, requires membrane proteins, and is stimulated by activators of G-proteins but not by Ca2+. The assay is simple, reliable, and rapid, and should permit the identification of proteins that are involved in the exocytotic fusion event.

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