Hyaluronic acid and the cumulus extracellular matrix induce increases in intracellular calcium in macaque sperm via the plasma membrane protein PH-20

Zygote ◽  
1999 ◽  
Vol 7 (3) ◽  
pp. 211-222 ◽  
Author(s):  
Gary N. Cherr ◽  
Ashley I. Yudin ◽  
Ming-Wen Li ◽  
Carol A. Vines ◽  
James W. Overstreet
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Hyaluronic Acid ◽  
Zona Pellucida ◽  
Cynomolgus Macaque ◽  
Fluorescein Isothiocyanate ◽  
Positive Control ◽  
Fab Fragments ◽  
Sperm Binding ◽  
Intracellular Ca

The hyaluronic acid (HA)-rich extracellular matrix (ECM) of the cumulus oophorus is known to facilitate fertilisation. It has been suggested that HA may enhance fertilisation in a number of species, and in macaque sperm, HA has been shown to increase the number of acrosome reactions that follow sperm binding to the zona pellucida. In this study, we investigated the effects of HA on intracellular Ca2+ in capacitated cynomolgus macaque sperm. Fluorometry studies using the intracellular Ca2+ indicator Fluo-3 showed that addition of 100 μg/ml of HA induced a rapid increase in intracellular Ca2+. This Ca2+ increase (approximately 2–3 times above basal levels) was inhibited by preincubation of sperm with Fab fragments of anti-recombinant PH-20 IgG. The frequency of acrosome reactions in sperm exposed to HA was not above control levels. A synthetic gel was prepared with similar viscosity to the cumulus and with HA trapped in its matrix. Video imaging of individual sperm was used to demonstrate that capacitated sperm swimming into the HA gel had increased intracellular Ca2+ levels. Preincubation of sperm with Fab fragments of anti-PH-20 IgG inhibited the increased intracellular Ca2+ levels induced by the HA gel. Sperm in control gel (no HA) did not show increased intracellular Ca2+, while sperm in gel containing anti-PH-20 IgG showed increased Ca2+ (positive control). Sperm loaded with Fluo-3 were allowed to interact with cynomologus macaque cumulus masses, and sperm within the cumulus ECM clearly showed increased intracellular Ca2+ that was inhibited when sperm were preincubated in anti-PH-20 Fab. Fluorescein isothiocyanate (FITC)-HA was found to bind to sperm over the acrosomal region, corresponding to PH-20 localisation, and this binding could be inhibited by preincubation of sperm with anti-PH-20 fragments. The results of this study show that HA increases intracellular Ca2+ in macaque sperm through interaction with plasma membrane PH-20. We propose that HA binding to plasma membrane PH-20 induces an aggregation of receptors that in turn results in intracellular signalling. As a result, sperm have higher basal CA2+ levels and are more responsive to induction of the acrosome reaction after binding to the zona pellucida.

Inhibition of sperm-egg fusion in the hamster and mouse by carbohydrates

Zygote ◽  
1994 ◽  
Vol 2 (3) ◽  
pp. 253-262 ◽  
Author(s):  
Ruben H. Ponce ◽  
Umbert A. Urch ◽  
Ryuzo Yanagimachi
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Zona Pellucida ◽  
Binding Proteins ◽  
Toxic Effects ◽  
Carbohydrate Binding ◽  
Dextran Sulphate ◽  
Membrane Glycoproteins ◽  
Carbohydrate Binding Proteins

SummaryAfter spermatozoa bind to and penetrate the extracellular matrix of the egg, the zona pellucida, they adhere to and fuse with the plasma membrane of the egg. Since sperm–egg fusion may involve membrane glycoproteins and/or carbohydrate binding proteins, we sought to test this hypothesis by challenging sperm–egg fusion in hamster and in mouse with added carbohydrates. In this study, a number of carbohydrate and glycoconjugates were examined for their ability to inhibit sperm–eggfusion. In the hamster, D(+)-glucosamine, D(+)-galactosamine, albumin-bovine-glucosamide and-galactosamide, fucoidan and dextran sulphate inhibited the fusion of spermatozoa with zona-free eggs. The same effects were seen in the mouse, except for the toxic effects of D(+)-galactosamine. These facts suggest a role of carbohydrate binding proteins or glycoproteins in the fertilisation process at the level of binding to and fusing with the oolemma.

Hyaluronic acid-dependent change in the extracellular matrix of mouse dermal fibroblasts that is conducive to cell proliferation

1988 ◽  
Vol 90 (2) ◽  
pp. 275-286 ◽  
Author(s):  
M. Yoneda ◽  
S. Shimizu ◽  
Y. Nishi ◽  
M. Yamagata ◽  
S. Suzuki ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Cell Proliferation ◽  
Hyaluronic Acid ◽  
Chondroitin Sulphate ◽  
Fluorescein Isothiocyanate ◽  
Dermal Fibroblasts ◽  
Fluorescent Label ◽  
Matrix Deposition ◽  
Chondroitin Sulphate Proteoglycans ◽  
Hyaluronic Acid Binding

Fluorescein isothiocyanate (FITC)-labelled hyaluronic acid, when incubated with subconfluent cultures of mouse dermal fibroblasts, was incorporated into the extracellular matrix. Deposition of the fluorescent label reached the maximum about 48 h after its addition. Hyaluronic acid decasaccharide, but not octasaccharide, inhibited the incorporation of the fluorescent label, suggesting that at least 10 sugar units in length are necessary for the incorporation of hyaluronic acid. A 2 M-urea extract of the cell layer had the ability to bind [3H]hyaluronic acid. Again, the binding was inhibited by hyaluronic acid decasaccharide but not by octasaccharide, suggesting the presence in the urea extract of a hyaluronic acid-binding molecule that may participate in the incorporation of hyaluronic acid. A supramolecular aggregate prepared by rate-zonal sedimentation from the 2 M-urea extract contained chondroitin sulphate proteoglycans capable of interacting with hyaluronic acid. Their core molecules were identical in size with those from a hyaluronic acid-binding chondroitin sulphate proteoglycan (PG-M) previously described in chick embryo fibroblasts. Immunofluorescence analyses with anti-proteoheparan sulphate antibodies indicated that both exogenous addition of hyaluronic acid and enhanced synthesis of hyaluronic acid caused a preferential decline in the proteoheparan sulphate level in the extracellular matrix. Subsequent to this change, the cells began transient DNA synthesis. We suggest that hyaluronic acid-dependent modulation of the level of proteoheparan sulphate in the extracellular matrix could be a causal event of cell proliferation.

Hyaluronic acid enhances induction of the acrosome reaction of human sperm through interaction with the PH-20 protein

Zygote ◽  
1998 ◽  
Vol 6 (2) ◽  
pp. 103-111 ◽  
Author(s):  
Khalida Sabeur ◽  
Gary N. Cherr ◽  
Ashley I. Yudin ◽  
James W. Overstreet
Keyword(s):  
Hyaluronic Acid ◽  
Intracellular Calcium ◽  
Direct Effect ◽  
Zona Pellucida ◽  
Acrosome Reaction ◽  
Human Sperm ◽  
Enhancing Effect ◽  
Fab Fragments ◽  
Progesterone Treatment ◽  
Data Support

When capacitated human sperm were treated with hyaluronic acid (HA) for 30 min prior to the addition of progesterone or solubilised human zonae pellucidae, there was a significant increase in the percentage of acrosome reactions. Progesterone treatment alone increased acrosome reactions from 10.5% to 21.8% and pretreatment with 100 µg/ml HA resulted in 33.0% acrosome reactions. With zonae pellucidae treatment alone the increase was from 9.0% to 23.5% and with HA pretreatment it was 48.8%. HA treatment alone had no direct effect on acrosome reactions, and the enhancing effect of HA was not removed when sperm were washed prior to the addition of either acrosome reaction agonist. Experiments with sperm 5 min after HA treatment demonstrated that enhancement of acrosome reactions was apparent as early as 1 min after addition of zonae and within 5 min after addition of progesterone. When sperm were pretreated with Fab fragments of anti-PH-20 IgG, then with HA and then with progesterone or zonae pellucidae, there was no enhancement of the acrosome reaction. Fab treatment did not induce acrosome reactions and did not interfere with the action of either agonist in the absence of HA. Sperm that were treated with HA had significantly higher intracellular calcium levels, and pretreatment with Fab reduced this increase to 42.7%. Addition of progesterone to HA-treated sperm was followed by another large increase in intracellular calcium, which was lower when sperm were pretreated with Fab. These results suggest that HA interacts with the PH-20 protein to increase basal levels of intracellular calcium and thereby potentiates the acrosome reaction. The data support the hypothesis that HA in the cumulus matrix may act to prime the fertilising sperm for induction of the acrosome reaction by constituents of the cumulus and/or zona pellucida.

scholarly journals Sperm IZUMO1 Is Required for Binding Preceding Fusion With Oolemma in Mice and Rats

2022 ◽  
Vol 9 ◽  
Author(s):  
Takafumi Matsumura ◽  
Taichi Noda ◽  
Yuhkoh Satouh ◽  
Akane Morohoshi ◽  
Shunsuke Yuri ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Zona Pellucida ◽  
Knockout Mice ◽  
Knockout Mouse ◽  
Acrosome Reaction ◽  
Male Rats ◽  
Sperm Protein ◽  
Complex Processes ◽  
Sperm Binding ◽  
Mammalian Spermatozoa

Fertilization occurs as the culmination of multi-step complex processes. First, mammalian spermatozoa undergo the acrosome reaction to become fusion-competent. Then, the acrosome-reacted spermatozoa penetrate the zona pellucida and adhere to and finally fuse with the egg plasma membrane. IZUMO1 is the first sperm protein proven to be essential for sperm-egg fusion in mammals, as Izumo1 knockout mouse spermatozoa adhere to but fail to fuse with the oolemma. However, the IZUMO1 function in other species remains largely unknown. Here, we generated Izumo1 knockout rats by CRISPR/Cas9 and found the male rats were infertile. Unlike in mice, Izumo1 knockout rat spermatozoa failed to bind to the oolemma. Further investigation revealed that the acrosome-intact sperm binding conceals a decreased number of the acrosome-reacted sperm bound to the oolemma in Izumo1 knockout mice. Of note, we could not see any apparent defects in the binding of the acrosome-reacted sperm to the oolemma in the mice lacking recently found fusion-indispensable genes, Fimp, Sof1, Spaca6, or Tmem95. Collectively, our data suggest that IZUMO1 is required for the sperm-oolemma binding prior to fusion at least in rat.

The extracellular matrix of echinoderm and amphibian eggs: Visualization in quick-frozen, deep-etched, and rotary-shadowed specimens

1990 ◽  
Vol 48 (3) ◽  
pp. 60-61
Author(s):  
Barry Bonnell ◽  
Carolyn Larabell ◽  
Douglas Chandler
Keyword(s):  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Sea Urchin ◽  
Crystalline Structure ◽  
Cortical Granule ◽  
Two Dimensional ◽  
Small Peptides ◽  
Deep Etching ◽  
Quick Freezing ◽  
Acrosomal Reaction

Eggs of many species including those of echinoderms, amphibians and mammals exhibit an extensive extracellular matrix (ECM) that is important both in the reception of sperm and in providing a block to polyspermy after fertilization.In sea urchin eggs there are two distinctive coats, the vitelline layer which contains glycoprotein sperm receptors and the jelly layer that contains fucose sulfate glycoconjugates which trigger the acrosomal reaction and small peptides which act as chemoattractants for sperm. The vitelline layer (VL), as visualized by quick-freezing, deep-etching, and rotary-shadowing (QFDE-RS), is a fishnet-like structure, anchored to the plasma membrane by short posts. Orbiting above the VL are horizontal filaments which are thought to anchor the thicker jelly layer to the egg. Upon fertilization, the VL elevates and is transformed by cortical granule secretions into the fertilization envelope (FE). The rounded casts of microvilli in the VL are transformed into angular peaks and the envelope becomes coated inside and out with sheets of paracrystalline protein having a quasi-two dimensional crystalline structure.

Differences between antigenic determinants of pig and cat zona pellucida proteins

Reproduction ◽  
2000 ◽  
pp. 15-23 ◽  
Author(s):  
K Jewgenow ◽  
M Rohleder ◽  
I Wegner
Keyword(s):  
In Vitro Fertilization ◽  
Zona Pellucida ◽  
Antigenic Determinants ◽  
Vitro Fertilization ◽  
Contraceptive Vaccine ◽  
Sperm Binding ◽  
Dependent Inhibition ◽  
Dose Dependent Inhibition ◽  
Dose Dependent

Despite many efforts, the control of reproduction in feral cat populations is still a problem in urban regions around the world. Immunocontraception is a promising approach; thus the present study examined the suitability of the widely used pig zona pellucida proteins (pZP) for contraception in feral domestic cats. Purified zona pellucida proteins obtained from pig and cat ovaries were used to produce highly specific antisera in rabbits. Antibodies against pZP raised in rabbits or lions were not effective inhibitors of either in vitro sperm binding (cat spermatozoa to cat oocytes) or in vitro fertilization in cats, whereas antibodies against feline zona pellucida proteins (fZP) raised in rabbits showed a dose-dependent inhibition of in vitro fertilization. Immunoelectrophoresis, ELISA and immunohistology of ovaries confirmed these results, showing crossreactivity of anti-fZP sera to fZP and to a lesser extent to pZP, but no interaction of anti-pZP sera with fZP. It is concluded that cat and pig zonae pellucidae express a very small number of shared antigenic determinants, making the use of pZP vaccine in cats questionable. A contraceptive vaccine based on feline zona pellucida determinants will be a better choice for the control of reproduction in feral cats if immunogenity can be achieved.

Interspecific variation of zona pellucida glycoconjugates in several species of marsupial

Reproduction ◽  
2000 ◽  
pp. 111-120 ◽  
Author(s):  
JA Chapman ◽  
OW Wiebkin ◽  
WG Breed
Keyword(s):  
Glycine Max ◽  
Sialic Acid ◽  
Zona Pellucida ◽  
Fluorescein Isothiocyanate ◽  
Interspecific Variation ◽  
Lectin Histochemistry ◽  
Sialic Acids ◽  
Arachis Hypogea ◽  
Marsupial Species ◽  
Similar Intensity

The zona pellucida glycoconjugate content of several marsupial species was investigated using differential lectin histochemistry. Ovaries from fat-tailed dunnarts, a southern brown bandicoot, grey short-tailed opossums, brushtail possums, ringtail possums, koalas and eastern grey kangaroos were fixed, embedded in paraffin wax, sectioned and stained with ten fluorescein isothiocyanate-conjugated lectins. Sections were also incubated with either neuraminidase or saponified, respectively, before incubation with the lectins to identify saccharide residues masked by sialic acids or O-acetyl groups on sialic acids. The zonae pellucidae surrounding the oocytes of the marsupials demonstrated interspecific variation in glycoconjugate content, with mannose-containing glycoconjugates exhibiting the greatest variation. Some of the zona pellucida glycoconjugates of all species, except those of the opossums, were masked by sialic acid with an increase in fluorescence with lectins from Arachis hypogea (PNA), and Glycine max (SBA), after desialylation. The disaccharide beta-galactose(1-4)N-acetyl-D-glucosamine appeared to be conformationally masked by O-acetyl groups of sialic acids in the zonae pellucidae of all species, with an increase in fluorescence with the lectin from Erythrina cristagalli (ECA), after saponification. Similar intensity and localization of beta-(1-4)-N-acetyl-D-glucosamine, as shown by staining of the lectin from Triticum vulgaris (WGA), to the inner and outer regions of the zona pellucida, were found to those reported in eutherian species. WGA fluorescence became uniform throughout the zonae pellucidae after saponification, indicating differential O-acetylation of sialic acids on the internal compartment of the zonae pellucidae.

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