Membrane fusion events in the Ca2+/ionophore-induced acrosome reaction of ram spermatozoa

1986 ◽  
Vol 81 (1) ◽  
pp. 43-63 ◽  
Author(s):  
J.E. Flechon ◽  
R.A. Harrison ◽  
B. Flechon ◽  
J. Escaig
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Morphological Changes ◽  
Anterior Part ◽  
Freeze Fracture ◽  
Calcium Ionophore ◽  
Limited Area ◽  
Ionophore A23187 ◽  
Early Effect ◽  
Effect Of Treatment

An acrosome reaction was induced in ejaculated ram spermatozoa by treatment with calcium and the ionophore A23187. Samples were fixed at different times after initiation of induction, and the morphological changes within the head membranes that took place as exocytosis occurred were studied in freeze-fracture replicas. Reacted acrosomes appeared in individual spermatozoa within the calcium/ionophore-treated population at different times after the start of treatment; the first cells had reacted by 10 min, whereas some took more than 40 min to react. No changes were observed in control populations. An early effect of treatment (seen in most cells within 10 min) was the appearance of particle-free ‘clearings’ in the plasma membrane over the entire acrosomal region, with aggregation of intramembranous particles between and around these ‘clearings’. At the same time, there was an increase in the number of large particles (greater than or equal to 10 nm) within the plasma membrane over the ‘lunula’ of the equatorial segment and the anterior part of the post-acrosomal region. Fusion of the plasma and outer acrosomal membranes began in a limited area at the border between the anterior and equatorial segments of the acrosome. It then spread, following arborescent pathways, sideways along this border and forwards towards the apex of the head. This labyrinthic propagation resulted in an ‘acrosomal cap’ increasingly fenestrated towards its posterior margin. Fusion propagation over the equatorial segment was inhibited, apparently as a result of the highly ordered structure of the membranes in this region.

Effects of hypothermic liquid storage and cryopreservation on basal and induced plasma membrane phospholipid disorder and acrosome exocytosis in boar spermatozoa

2005 ◽  
Vol 17 (4) ◽  
pp. 467 ◽  
Author(s):  
H. D. Guthrie ◽  
G. R. Welch
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Fluorescein Isothiocyanate ◽  
Calcium Ionophore A23187 ◽  
Membrane Phospholipid ◽  
Ionophore A23187 ◽  
Liquid Storage ◽  
Boar Spermatozoa ◽  
Plasma Membrane Phospholipid

Flow cytometry was utilised to determine whether short-term (Day 1) or long-term hypothermic liquid storage (Day 5), or cryopreservation of boar spermatozoa (1) caused changes in plasma membrane phospholipid disorder (MPLD) and acrosome exocytosis (AE), indicative of an advanced stage of capacitation or acrosome status, and (2) facilitated or inhibited the induction of capacitation and the acrosome reaction. Merocyanine with Yo-Pro-1 and peanut agglutinin–fluorescein isothiocyanate with propidium iodide were used to identify MPLD and AE, respectively, in viable spermatozoa. The incidence of basal sperm MPLD and AE in fresh semen was very low (1.1 and 2.2%, respectively) and was increased (P < 0.05) only a small amount in Day 5 and cryopreserved semen (3–8%). Compared to no bicarbonate, incubation with bicarbonate increased MPLD, but the response was greatest (P < 0.05) in fresh sperm (52.3%) compared with Day 1 (36.6%), Day 5 (13.9%) and cryopreserved sperm (13.6%). Incubation with calcium ionophore A23187 increased AE in spermatozoa, but the response was less (P < 0.05) for fresh (34%) and cryopreserved (27%) semen than for Day 1 (45%) and Day 5 (57%) semen. In summary, hypothermic liquid storage and cryopreservation of boar spermatozoa did not advance capacitation or acrosome status in viable spermatozoa, but did alter their responses to induction of capacitation and the acrosome reaction.

Growth factors and the primary cilium in BALB/c 3T3 cells

1987 ◽  
Vol 45 ◽  
pp. 830-831
Author(s):  
R. W. Tucker ◽  
N. S. More ◽  
S. Jayaraman
Keyword(s):  
Growth Factors ◽  
Primary Cilium ◽  
Cultured Cells ◽  
Morphological Changes ◽  
Calcium Ionophore ◽  
Growth Stimulation ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
3T3 Cells ◽  
Microtubule Depolymerization

The mechanisms by which polypeptide growth factors Induce DNA synthesis in cultured cells is not understood, but morphological changes Induced by growth factors have been used as clues to Intracellular messengers responsible for growth stimulation. One such morphological change has been the transient disappearance of the primary cilium, a “9 + 0” cilium formed by the perinuclear centriole in interphase cells. Since calcium ionophore A23187 also produced both mitogenesis and ciliary changes, microtubule depolymerization might explain ciliary disappearance monitored by indirect immunofluorescence with anti-tubulin antibody. However, complete resorption and subsequent reformation of the primary cilium occurs at mitosis, and might also account for ciliary disappearance induced by growth factors. To settle this issue, we investigated the ultrastructure of the primary cilium using serial thin-section electron microscopy of quiescent BALB/c 3T3 cells before and after stimulation with serum.

Acrosome stability in the spermatozoa of dasyurid marsupials

2008 ◽  
Vol 20 (2) ◽  
pp. 295 ◽  
Author(s):  
N. A. Czarny ◽  
K. E. Mate ◽  
J. C. Rodger
Keyword(s):  
Disulfide Bonds ◽  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Macropus Eugenii ◽  
Sminthopsis Crassicaudata ◽  
Triton X ◽  
Physical And Chemical ◽  
Physical Challenge

The spermatozoa of most marsupials lack nuclear stabilising disulfide-bonded protamines found in eutherian mammals. However, disulfide stabilisation has been observed in the acrosome of macropodid (Macropus eugenii) and phalangerid (Trichosurus vulpecula) marsupials. As a result this organelle, which is normally fragile in eutherian mammals, is robust and able to withstand physical and chemical challenge in these marsupials. The present study examined acrosomal characteristics of the spermatozoa of three dasyurid marsupials; the fat-tailed dunnart (Sminthopsis crassicaudata), eastern quoll (Dasyurus viverrinus) and northern quoll (Dasyurus hallucatus). In all species examined Bryan’s staining demonstrated that significant acrosomal loss occurred following physical challenge with osmotic stress, cryopreservation without cryoprotectant and exposure to detergent (Triton-X). Bromobimane staining indicated that the acrosomes of dasyurids lacked stabilising disulfide bonds. As reported for the wallaby and possum, calcium ionophore (A23187) did not induce the acrosome reaction-like exocytosis in dasyurid spermatozoa but treatment with diacylglycerol (DiC8) caused significant acrosome loss at concentrations similar to those effective for other marsupials. The present study found that the spermatozoa of dasyurids are more sensitive to physical challenge than the previously-studied marsupials and we suggest that this is due to the absence of acrosomal stabilising disulfide bonds.

scholarly journals Bryostatin 1 induces differentiation of B-chronic lymphocytic leukemia cells

Blood ◽  
1989 ◽  
Vol 74 (5) ◽  
pp. 1747-1757 ◽  
Author(s):  
HG Drexler ◽  
SM Gignac ◽  
RA Jones ◽  
CS Scott ◽  
GR Pettit ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Rna Synthesis ◽  
Morphological Changes ◽  
Marine Invertebrate ◽  
Qualitative Difference ◽  
Calcium Ionophore ◽  
Lymphocytic Leukemia ◽  
Ionophore A23187 ◽  
Bryostatin 1 ◽  
Stimulatory Action

Abstract Peripheral blood cells from nine patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with bryostatin 1 (a macrocyclic lactone derived from a marine invertebrate). Like the phorbol ester 12- 0-tetradecanoyl-phorbol 13-acetate (TPA), bryostatin 1 activates protein kinase C (PKC), which plays a central role in the phosphatidylinositol signal transduction pathway. The effects of bryostatin 1 alone and in combination with TPA or with the calcium mobilizing ionophore A23187 were assessed by morphological appearance, cell adherence and aggregation, RNA and DNA synthesis, and immunoglobulin (Ig) production. While eight of nine B-CLL cultures remained proliferatively inert, bryostatin 1 could effectively trigger activation and differentiation of B-CLL cells in all cases as inferred by the induction of morphological changes, RNA synthesis, and monotypic Ig production. Addition of calcium ionophore A23187 to bryostatin 1- exposed cells resulted in significantly increased values for RNA synthesis and Ig production and in the acquisition of plasmacytoid morphology. Bryostatin 1 and the dual signal of bryostatin 1 plus A23187 mimicked the stimulatory action of TPA and the combination of TPA plus A23187, respectively. Overall, bryostatin 1 was less active than equivalent concentrations of TPA. This lesser efficacy may, however, reflect a quantitative rather than qualitative difference. Bryostatin 1 partially antagonized TPA-mediated effects on B-CLL cells suggesting different modes of action by the two activators. These studies indicate that bryostatin 1 has effective differentiation- inducing properties on B-CLL cells that can differentiation-inducing properties on B-CLL cells that can be accentuated by a calcium ionophore.

Patterns of calcium localization in pancreatic endocrine cells

1976 ◽  
Vol 21 (1) ◽  
pp. 107-117
Author(s):  
M. Ravazzola ◽  
F. Malaisse-Lagae ◽  
M. Amherdt ◽  
A. Perrelet ◽  
W.J. Malaisse ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
B Cells ◽  
Endocrine Cells ◽  
Secretory Granules ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Calcium Localization ◽  
A Cells ◽  
D Cells

Subcellular calcium localization in the dndocrine cells of rat pancreas was studied by the pyroantimonate precipitation technique. Calcium-containing electron-dense deposits in the endocrine cells were mostly found within secretory granules and along the plasma membrane, but their pattern of distribution in A-, B- and D-cells displayed qualitative and quantitative differences. In B-cells, numerous secretory granules contained deposits located in the halo surrounding the granule core. In A-cells, only few granules contained precipitates in their halo, whereas in D-cells, deposits were situated in the dense core of the secretory granules. Deposits along the plasma membrane occurred generally on the outer leaflet of the plasma membrane of B- and D-cells and on the inner leaflet of that of A-cells. In islets incubated at a high glucose concentration or in the presence of the calcium ionophore A23187, the number of beta granules containing precipitates was significantly increased. By contrast, only few deposits were observed in B-cells incubated in calcium-deprived medium enriched with EGTA. These findings indicate that: the pattern of calcium localization varies in different islet cell types; in B-cells the secretory granules represent one of the major stores of intracellular calcium; and that this store undergoes changes in conditions which alter insulin release.

The activation of proteolysis in the acrosome reaction of guinea-pig sperm

1978 ◽  
Vol 32 (1) ◽  
pp. 153-164
Author(s):  
D.P. Green
Keyword(s):  
Guinea Pig ◽  
Acrosome Reaction ◽  
Morphological Changes ◽  
Divalent Metal ◽  
Free Calcium ◽  
Ionophore A23187 ◽  
Reaction Proceeds ◽  
Insoluble Form ◽  
Acrosin Activity ◽  
Sperm Population

The divalent metal cation ionophore A23187 rapidly induces a normal acrosome reaction in a population of guinea-pig sperm suspended in calcium medium. In the course of the acrosome reaction, proacrosin, the zymogen precursor of the protease acrosin, is activated. Although the acrosome reaction causes exocytosis of the acrosomal contents, ‘soluble’ acrosin is not released in significant amounts until well after the sperm population as a whole has undergone an acrosome reaction. This suggests that proacrosin is stored within the acrosome in an insoluble form and that exocytosis of the acrosomal contents in the acrosome reaction is insufficient, by itself, to cause its immediate dissolution. Electron micrographs of sperm undergoing an A23187-induced acrosome reaction in the presence of the acrosin inhibitors benzamidine, p-amino-benzamidine and phenylmethylsulphonyl fluoride show that the acrosome reaction proceeds normally but that dispersal of the acrosomal contents is inhibited. These morphological changes are, for the most part, below the limit of resolution of the light microscope and using light microscopy to assess whether an acrosome reaction has taken place, it can be mistakenly inferred that the reaction itself is inhibited by the acrosin inhibitors. The inhibition of the dispersal of the acrosomal contents by acrosin inhibitors suggests that acrosin activity is important in solubilizing acrosin. These experimental observations, taken with the evidence that the acrosome reaction is a response to an increase in intracellular free calcium, have been taken as the basis of a proposal for the mechanism of proacrosin activation in the acrosome reaction.

Capacity of goat epididymal spermatozoa to undergo the acrosome reaction and subsequent fusion with the egg plasma membrane

1993 ◽  
Vol 5 (3) ◽  
pp. 239 ◽  
Author(s):  
H Harayama ◽  
H Kusunoki ◽  
S Kato
Keyword(s):  
Plasma Membrane ◽  
Acrosome Reaction ◽  
Morphological Changes ◽  
Glass Tube ◽  
Electron Microscopic Examination ◽  
Electron Microscopic ◽  
Acrosomal Membrane ◽  
Sperm Penetration ◽  
Epididymal Spermatozoa ◽  
Caput Epididymidis

The capacity to undergo the acrosome reaction and subsequent fusion with the egg plasma membrane was examined in goat epididymal spermatozoa. Spermatozoa from the proximal and distal caput and distal cauda were preincubated in a sealed glass tube for induction of the acrosome reaction, and their viability, acrosome morphology and penetrability into zona-free hamster eggs were determined. A simplified triple-stain technique revealed that most of the preincubated live spermatozoa in the samples from the distal caput and distal cauda epididymides underwent morphological changes that indicated the occurrence of the acrosome reaction. Electron microscopic examination revealed that the outer acrosomal membrane of many spermatozoa in these samples showed fusion at multiple sites to the plasma membrane. However, the rates of acrosome-reacted cells in the proximal caput spermatozoa were still lower. The sperm penetration assay demonstrated that the penetration rates of distal caput and distal cauda spermatozoa preincubated for 2 h were 93% and 74% respectively, whereas proximal caput spermatozoa scarcely penetrated into eggs. These results indicate that increasing numbers of goat spermatozoa improve in the functions related to the acrosome reaction and subsequent fusion with the egg plasma membrane during their transit through the caput epididymidis.

Relocation of myosin and actin, kinesin and tubulin in the acrosome reaction of bovine spermatozoa

2009 ◽  
Vol 21 (2) ◽  
pp. 364 ◽  
Author(s):  
Ifigenia Oikonomopoulou ◽  
Hitesh Patel ◽  
Paul F. Watson ◽  
Peter D. Chantler
Keyword(s):  
Molecular Motors ◽  
Acrosome Reaction ◽  
Molecular Motor ◽  
Calcium Ionophore ◽  
Cell Types ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Sequence Of Events ◽  
Bovine Spermatozoa ◽  
Number Of Cells

The mammalian acrosome reaction is a specialised exocytotic event. Although molecular motors are known to be involved in exocytosis in many cell types, their potential involvement in the acrosome reaction has remained unknown. Here, it has been shown that actin is localised within the equatorial segment and in the marginal acrosomal ridge of the heads of unreacted bull spermatozoa. Myosins IIA and IIB are found within the anterior acrosomal margins of virtually all sperm cells and, less prominently, within the equatorial segment. Tubulin was detected in the equatorial segment and around the periphery of the acrosome while kinesin was prominent in the equatorial segment. After induction of the acrosome reaction by means of the calcium ionophore A23187, the number of cells exhibiting actin fluorescence intensity in the anterior acrosomal margin decreased four-fold and those displaying equatorial segment fluorescence decreased 3.5-fold; myosin IIA immunofluorescence decreased in intensity with most spermatozoa losing equatorial staining, whereas there was little change in the distribution or intensity of myosin IIB immunofluorescence, except for a ~20% decrease in the number of cells exhibiting acrosomal staining. Tubulin became largely undetectable within the head and kinesin staining spread rostrally over the main acrosome region. A possible sequence of events that ties in these observations of molecular motor involvement with the known participation of SNARE proteins is provided.

Ca2+ regulation of tight-junction permeability and structure in Necturus gallbladder

1983 ◽  
Vol 245 (3) ◽  
pp. C203-C212 ◽  
Author(s):  
C. E. Palant ◽  
M. E. Duffey ◽  
B. K. Mookerjee ◽  
S. Ho ◽  
C. J. Bentzel
Keyword(s):  
Cell Membrane ◽  
Tight Junction ◽  
Morphological Changes ◽  
Freeze Fracture ◽  
Transepithelial Resistance ◽  
Mucosal Cell ◽  
Base Line ◽  
Ionophore A23187 ◽  
Necturus Gallbladder ◽  
Tight Junction Permeability

To explore the role of Ca2+ in tight-junction permeability, the Necturus gallbladder was exposed to varying Ca2+ concentrations and to the Ca2+ ionophore A23187 added to the mucosal side (1.9 X 10(-6) to 6.8 X 10(-5) M). Electrophysiological parameters measured in an Ussing-type chamber were correlated with tight-junction morphology revealed by freeze-fracture electron microscopy. In Ca2+-free bathing media, transepithelial resistance decreases and tight-junctional ultrastructure is fragmented. In 1.8 mM Ca2+ media, A23187 induces an initial drop in transepithelial resistance, followed by an increase in transepithelial resistance to a value 20% above base line. At peak response to A23187, NaCl diffusion potentials decrease. Freeze-fracture replicas reveal that the number of junctional strands increase pari passu with junctional depth. Both physiological and morphological changes were partially reversible. The initial decrease in transepithelial resistance coincided with a persistent hyperpolarization of the mucosal cell membrane potential difference and a decrease in the mucosal-to-serosal cell membrane resistance ratio. Thus A23187 alters both the transcellular and paracellular pathway, resulting in opposing effects on transepithelial resistance.

Leptin is involved in acrosome reaction by facilitating activation of MAPK cascades in the Chinese mitten crab, Eriocheir sinensis

2020 ◽  
Vol 70 (1) ◽  
pp. 81-95
Author(s):  
Qing Li ◽  
Haitao Zhao ◽  
Lin He ◽  
Hongdan Yang ◽  
Qun Wang
Keyword(s):  
Acrosome Reaction ◽  
Calcium Ionophore ◽  
Eriocheir Sinensis ◽  
Calcium Ionophore A23187 ◽  
Mitogen Activated Protein Kinase ◽  
Ionophore A23187 ◽  
Mapk Cascades ◽  
Mitten Crab ◽  
Mitogen Activated Protein

Abstract The role of leptin has been documented in several studies, including activated threonine phosphorylation of extracellular signal-regulated kinase (ERK1/2) in the reproduction of rodents and humans. Our previous studies have demonstrated that mitogen-activated protein kinase (MAPK) cascades ERK, P38, and c-Jun N-terminal kinase (JNK) are involved in the spermatogenesis and acrosome reaction of Eriocheir sinensis. Therefore, the aim of this study was to investigate the expression of leptin and its receptor (LepR), and the effect of leptin on MAPK cascades during calcium ionophore A23187-induced spermatozoa acrosome reaction in crabs. Successful western blotting revealed a 16 kDa band for leptin, and 120 kDa and 90 kDa bands for the obese receptor (LepR), respectively, in the tested male reproductive tissues. Both leptin and LepR were localized at the pro-acrosomal vesicle and apical cap (AC) of spermatids, suggesting their role in the subsequent acrosome reaction. Moreover, acrosome reaction can be enhanced by leptin, and this effect decreased due to the anti-LepR antibody. Afterwards, we investigated the effects of leptin on MAPK cascades. The results showed that leptin mainly activated the phosphorylation of ERK, P38 and JNK proteins in the apical cap during the acrosome reaction in crab spermatozoa. This study addresses the role of leptin on spermatozoa, and suggests that leptin may induce molecular changes associated with spermatozoa during acrosome reaction.

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