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Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

Zimmerman SW, Manandhar G, Yi YJ, Gupta SK, Sutovsky M, Odhiambo JF, Powell MD, Miller DJ, Sutovsky P - PLoS ONE (2011)

Bottom Line: ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis.Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates.This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

View Article: PubMed Central - PubMed

Affiliation: Division of Animal Science, and Departments of Obstetrics, Gynecology, and Women's Health, University of Missouri-Columbia, Columbia, Missouri, United States of America.

ABSTRACT
Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

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Related in: MedlinePlus

The ZPP-induced formation, detachment and breakdown of acrosomal shrouds traced by flow cytometry and epifluorescence microscopy with lectin PNA-FITC.Lectin PNA binds to terminal β-galactose residues of disaccharides present on the outer acrosomal membrane (OAM), which is concealed in the non-capacitated spermatozoa, partially exposed in the capacitated ones and completely exposed during acrosomal exocytosis, which encompasses the vesiculation of OAM, and formation and eventual breakdown of the acrosomal shroud. During fertilization, the shroud remains attached to egg coat surface, forming a viscous cloud around the penetrating sperm head [1]. In a cell free system with live sperm and soluble ZPP, the shrouds eventually detach from the sperm heads of capacitated, ZPP-exposed spermatozoa and disintegrate, unless proteasomal inhibitors are included in coincubation mixture. The flow cytometric histograms of 5,000 cells/events per sample show an average from two replicates for the percentage of highly fluorescent cells (%-value; mean±SE) corresponding to the shadowed area of the histogram, and the median fluorescence of all flow cytometric events in the entire sample (M-value; mean±SE). Histograms of relative PNA-fluorescence are shown separately for gated acrosomal shrouds detached from sperm cells (left histogram column) and for the entire sample, including sperm cells and detached acrosomal shrouds (right histogram column). (a) The relative fluorescence of PNA is low prior to capacitation, in flow cytometer and by epifluorescence, because the OAM is concealed under sperm plasma membrane and not exposed for PNA-binding. (b) Fluorescence increases during capitation because OAM becomes exposed in the capacitated spermatozoa [2]; yet other spermatozoa undergo spontaneous acrosomal exocytosis. (c) Fluorescence level retreats back after sperm-ZPP coincubation under control conditions (only vehicles for proteasomal inhibitors were added) because the acrosomal shrouds detach from the spermatozoa and disintegrate. Most spermatozoa in this treatment show only the residual PNA binding to the newly exposed inner acrosomal membrane of the exocytosed spermatozoa (arrowheads); other spermatozoa are still in the process of exocytosis (arrow). (d) In the inhibitor group, fluorescence is even higher than in capacitated spermatozoa because proteasomal inhibition prevents the disintegration of acrosomal shrouds, whether still attached to sperm heads (arrow) or detached (arrowheads).
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pone-0017256-g003: The ZPP-induced formation, detachment and breakdown of acrosomal shrouds traced by flow cytometry and epifluorescence microscopy with lectin PNA-FITC.Lectin PNA binds to terminal β-galactose residues of disaccharides present on the outer acrosomal membrane (OAM), which is concealed in the non-capacitated spermatozoa, partially exposed in the capacitated ones and completely exposed during acrosomal exocytosis, which encompasses the vesiculation of OAM, and formation and eventual breakdown of the acrosomal shroud. During fertilization, the shroud remains attached to egg coat surface, forming a viscous cloud around the penetrating sperm head [1]. In a cell free system with live sperm and soluble ZPP, the shrouds eventually detach from the sperm heads of capacitated, ZPP-exposed spermatozoa and disintegrate, unless proteasomal inhibitors are included in coincubation mixture. The flow cytometric histograms of 5,000 cells/events per sample show an average from two replicates for the percentage of highly fluorescent cells (%-value; mean±SE) corresponding to the shadowed area of the histogram, and the median fluorescence of all flow cytometric events in the entire sample (M-value; mean±SE). Histograms of relative PNA-fluorescence are shown separately for gated acrosomal shrouds detached from sperm cells (left histogram column) and for the entire sample, including sperm cells and detached acrosomal shrouds (right histogram column). (a) The relative fluorescence of PNA is low prior to capacitation, in flow cytometer and by epifluorescence, because the OAM is concealed under sperm plasma membrane and not exposed for PNA-binding. (b) Fluorescence increases during capitation because OAM becomes exposed in the capacitated spermatozoa [2]; yet other spermatozoa undergo spontaneous acrosomal exocytosis. (c) Fluorescence level retreats back after sperm-ZPP coincubation under control conditions (only vehicles for proteasomal inhibitors were added) because the acrosomal shrouds detach from the spermatozoa and disintegrate. Most spermatozoa in this treatment show only the residual PNA binding to the newly exposed inner acrosomal membrane of the exocytosed spermatozoa (arrowheads); other spermatozoa are still in the process of exocytosis (arrow). (d) In the inhibitor group, fluorescence is even higher than in capacitated spermatozoa because proteasomal inhibition prevents the disintegration of acrosomal shrouds, whether still attached to sperm heads (arrow) or detached (arrowheads).

Mentions: Binding of soluble ZPC protein to capacitated spermatozoa in our co-incubation system was readily detectable by immunofluorescence with antibodies specific to porcine sperm receptor glycoprotein ZPC (Fig. 2a). This binding was not affected by proteasomal inhibitors, yet the proteasomal inhibitors altered the ZP-protein-induced AE and resulted in the retention of the acrosomal shrouds on many spermatozoa (Fig. 2a). The observation that the inclusion of proteasomal inhibitors prevented the detachment and/or the disintegration of acrosomal shrouds from the ZPP-exposed sperm heads was further supported by the experiments showing lesser presence of acrosome-derived proteasomes in the supernatants in sperm-ZPP fractions coincubated in the presence of proteasomal inhibitors (Fig. 2b). Sperm acrosomal status and formation of the acrosomal shrouds upon co-incubation were monitored by flow cytometry of live, ZPP-exposed and control spermatozoa in which the acrosomes were labeled with fluorescently-conjugated, outer acrosomal membrane-binding lectin PNA [23] (Fig. 2c-e). Patterns of acrosomal shroud labeling with PNA and their flow cytometry-measured fluorescence intensities are shown in Fig. 3 a-d.


Sperm proteasomes degrade sperm receptor on the egg zona pellucida during mammalian fertilization.

Zimmerman SW, Manandhar G, Yi YJ, Gupta SK, Sutovsky M, Odhiambo JF, Powell MD, Miller DJ, Sutovsky P - PLoS ONE (2011)

The ZPP-induced formation, detachment and breakdown of acrosomal shrouds traced by flow cytometry and epifluorescence microscopy with lectin PNA-FITC.Lectin PNA binds to terminal β-galactose residues of disaccharides present on the outer acrosomal membrane (OAM), which is concealed in the non-capacitated spermatozoa, partially exposed in the capacitated ones and completely exposed during acrosomal exocytosis, which encompasses the vesiculation of OAM, and formation and eventual breakdown of the acrosomal shroud. During fertilization, the shroud remains attached to egg coat surface, forming a viscous cloud around the penetrating sperm head [1]. In a cell free system with live sperm and soluble ZPP, the shrouds eventually detach from the sperm heads of capacitated, ZPP-exposed spermatozoa and disintegrate, unless proteasomal inhibitors are included in coincubation mixture. The flow cytometric histograms of 5,000 cells/events per sample show an average from two replicates for the percentage of highly fluorescent cells (%-value; mean±SE) corresponding to the shadowed area of the histogram, and the median fluorescence of all flow cytometric events in the entire sample (M-value; mean±SE). Histograms of relative PNA-fluorescence are shown separately for gated acrosomal shrouds detached from sperm cells (left histogram column) and for the entire sample, including sperm cells and detached acrosomal shrouds (right histogram column). (a) The relative fluorescence of PNA is low prior to capacitation, in flow cytometer and by epifluorescence, because the OAM is concealed under sperm plasma membrane and not exposed for PNA-binding. (b) Fluorescence increases during capitation because OAM becomes exposed in the capacitated spermatozoa [2]; yet other spermatozoa undergo spontaneous acrosomal exocytosis. (c) Fluorescence level retreats back after sperm-ZPP coincubation under control conditions (only vehicles for proteasomal inhibitors were added) because the acrosomal shrouds detach from the spermatozoa and disintegrate. Most spermatozoa in this treatment show only the residual PNA binding to the newly exposed inner acrosomal membrane of the exocytosed spermatozoa (arrowheads); other spermatozoa are still in the process of exocytosis (arrow). (d) In the inhibitor group, fluorescence is even higher than in capacitated spermatozoa because proteasomal inhibition prevents the disintegration of acrosomal shrouds, whether still attached to sperm heads (arrow) or detached (arrowheads).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044170&req=5

pone-0017256-g003: The ZPP-induced formation, detachment and breakdown of acrosomal shrouds traced by flow cytometry and epifluorescence microscopy with lectin PNA-FITC.Lectin PNA binds to terminal β-galactose residues of disaccharides present on the outer acrosomal membrane (OAM), which is concealed in the non-capacitated spermatozoa, partially exposed in the capacitated ones and completely exposed during acrosomal exocytosis, which encompasses the vesiculation of OAM, and formation and eventual breakdown of the acrosomal shroud. During fertilization, the shroud remains attached to egg coat surface, forming a viscous cloud around the penetrating sperm head [1]. In a cell free system with live sperm and soluble ZPP, the shrouds eventually detach from the sperm heads of capacitated, ZPP-exposed spermatozoa and disintegrate, unless proteasomal inhibitors are included in coincubation mixture. The flow cytometric histograms of 5,000 cells/events per sample show an average from two replicates for the percentage of highly fluorescent cells (%-value; mean±SE) corresponding to the shadowed area of the histogram, and the median fluorescence of all flow cytometric events in the entire sample (M-value; mean±SE). Histograms of relative PNA-fluorescence are shown separately for gated acrosomal shrouds detached from sperm cells (left histogram column) and for the entire sample, including sperm cells and detached acrosomal shrouds (right histogram column). (a) The relative fluorescence of PNA is low prior to capacitation, in flow cytometer and by epifluorescence, because the OAM is concealed under sperm plasma membrane and not exposed for PNA-binding. (b) Fluorescence increases during capitation because OAM becomes exposed in the capacitated spermatozoa [2]; yet other spermatozoa undergo spontaneous acrosomal exocytosis. (c) Fluorescence level retreats back after sperm-ZPP coincubation under control conditions (only vehicles for proteasomal inhibitors were added) because the acrosomal shrouds detach from the spermatozoa and disintegrate. Most spermatozoa in this treatment show only the residual PNA binding to the newly exposed inner acrosomal membrane of the exocytosed spermatozoa (arrowheads); other spermatozoa are still in the process of exocytosis (arrow). (d) In the inhibitor group, fluorescence is even higher than in capacitated spermatozoa because proteasomal inhibition prevents the disintegration of acrosomal shrouds, whether still attached to sperm heads (arrow) or detached (arrowheads).
Mentions: Binding of soluble ZPC protein to capacitated spermatozoa in our co-incubation system was readily detectable by immunofluorescence with antibodies specific to porcine sperm receptor glycoprotein ZPC (Fig. 2a). This binding was not affected by proteasomal inhibitors, yet the proteasomal inhibitors altered the ZP-protein-induced AE and resulted in the retention of the acrosomal shrouds on many spermatozoa (Fig. 2a). The observation that the inclusion of proteasomal inhibitors prevented the detachment and/or the disintegration of acrosomal shrouds from the ZPP-exposed sperm heads was further supported by the experiments showing lesser presence of acrosome-derived proteasomes in the supernatants in sperm-ZPP fractions coincubated in the presence of proteasomal inhibitors (Fig. 2b). Sperm acrosomal status and formation of the acrosomal shrouds upon co-incubation were monitored by flow cytometry of live, ZPP-exposed and control spermatozoa in which the acrosomes were labeled with fluorescently-conjugated, outer acrosomal membrane-binding lectin PNA [23] (Fig. 2c-e). Patterns of acrosomal shroud labeling with PNA and their flow cytometry-measured fluorescence intensities are shown in Fig. 3 a-d.

Bottom Line: ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis.Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates.This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

View Article: PubMed Central - PubMed

Affiliation: Division of Animal Science, and Departments of Obstetrics, Gynecology, and Women's Health, University of Missouri-Columbia, Columbia, Missouri, United States of America.

ABSTRACT
Despite decades of research, the mechanism by which the fertilizing spermatozoon penetrates the mammalian vitelline membrane, the zona pellucida (ZP) remains one of the unexplained fundamental events of human/mammalian development. Evidence has been accumulating in support of the 26S proteasome as a candidate for echinoderm, ascidian and mammalian egg coat lysin. Monitoring ZP protein degradation by sperm during fertilization is nearly impossible because those few spermatozoa that penetrate the ZP leave behind a virtually untraceable residue of degraded proteins. We have overcome this hurdle by designing an experimentally consistent in vitro system in which live boar spermatozoa are co-incubated with ZP-proteins (ZPP) solubilized from porcine oocytes. Using this assay, mimicking sperm-egg interactions, we demonstrate that the sperm-borne proteasomes can degrade the sperm receptor protein ZPC. Upon coincubation with motile spermatozoa, the solubilized ZPP, which appear to be ubiquitinated, adhered to sperm acrosomal caps and induced acrosomal exocytosis/formation of the acrosomal shroud. The degradation of the sperm receptor protein ZPC was assessed by Western blotting band-densitometry and proteomics. A nearly identical pattern of sperm receptor degradation, evident already within the first 5 min of coincubation, was observed when the spermatozoa were replaced with the isolated, enzymatically active, sperm-derived proteasomes. ZPC degradation was blocked by proteasomal inhibitors and accelerated by ubiquitin-aldehyde(UBAL), a modified ubiquitin protein that stimulates proteasomal proteolysis. Such a degradation pattern of ZPC is consistent with in vitro fertilization studies, in which proteasomal inhibitors completely blocked fertilization, and UBAL increased fertilization and polyspermy rates. Preincubation of intact zona-enclosed ova with isolated active sperm proteasomes caused digestion, abrasions and loosening of the exposed zonae, and significantly reduced the fertilization/polyspermy rates after IVF, accompanied by en-mass detachment of zona bound sperm. Thus, the sperm borne 26S proteasome is a candidate zona lysin in mammals. This new paradigm has implications for contraception and assisted reproductive technologies in humans, as well as animals.

Show MeSH
Related in: MedlinePlus