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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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Soluble ZPC protein binds to capacitated spermatozoa and triggers acrosomal exocytosis and separation of the acrosomal shrouds.(a). Immunofluorescence of sperm receptor protein ZPC (red) immediately after ZPP-sperm mixing (left), after 2 h of incubation (center; note the detached acrosomal shrouds-arrowheads) and after 2 h incubation with proteasomal inhibitors (right; arrows point to sperm with attached shrouds). (b). Addition of proteasomal inhibitors to sperm-ZPP coincubation limited the release of acrosomal proteasomes. Supernatant fractions (left panel) were collected from 10,000 sperm prior to mixing with ZPP (lane 1), after 2 h of coincubation (lane 2; appropriate vehicles were present), after 2 h co-incubation with proteasomal inhibitors added (lane 3) and immediately after sperm-ZPP mixing. Proteasomes were detected with a monoclonal antibody against alpha-type 20S proteasomal core subunits. The right panel shows the corresponding residual PAGE gel after protein transfer, confirming comparable protein loads between vehicle and inhibitor lanes (lanes 2&3). Lanes 1 and 4 contain only a small amount of protein because of limited acrosomal exocytosis. (c-e) Sperm acrosomal status and the formation and detachment of acrosomal shrouds upon co-incubation were monitored by flow cytometry of live spermatozoa in which the acrosomes were labeled with fluorescently-conjugated lectin PNA. (c) Gating of detached acrosomal shrouds (red dots) from spermatozoa (green dots) in scatter diagrams from flow cytometry of sperm-ZPP fractions. Each dot represents one flow cytometric event, a shroud or a sperm cell (2,000 events/fraction). (d) Number of intact acrosomal shrouds, gated in visible light scatter, is increased by addition of proteasomal inhibitors (lower right panel) to coincubation, compared to control sperm-ZPP fraction (upper right), and sperm fractions prior to (upper left) and after capacitation (upper right), not exposed to ZPP. A degree of spontaneous acrosomal exocytosis is expected during capacitation. (e) Percentage of detached acrosomal shrouds in the coincubation fraction (i.e. ratio of red dot-events to all events in the scatter diagrams gated on acrosomal shrouds), was increased significantly (ANOVA; p<0.05) by the addition of proteasomal inhibitor cocktail for the duration of sperm-ZPP coincubation.
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pone-0017256-g002: Soluble ZPC protein binds to capacitated spermatozoa and triggers acrosomal exocytosis and separation of the acrosomal shrouds.(a). Immunofluorescence of sperm receptor protein ZPC (red) immediately after ZPP-sperm mixing (left), after 2 h of incubation (center; note the detached acrosomal shrouds-arrowheads) and after 2 h incubation with proteasomal inhibitors (right; arrows point to sperm with attached shrouds). (b). Addition of proteasomal inhibitors to sperm-ZPP coincubation limited the release of acrosomal proteasomes. Supernatant fractions (left panel) were collected from 10,000 sperm prior to mixing with ZPP (lane 1), after 2 h of coincubation (lane 2; appropriate vehicles were present), after 2 h co-incubation with proteasomal inhibitors added (lane 3) and immediately after sperm-ZPP mixing. Proteasomes were detected with a monoclonal antibody against alpha-type 20S proteasomal core subunits. The right panel shows the corresponding residual PAGE gel after protein transfer, confirming comparable protein loads between vehicle and inhibitor lanes (lanes 2&3). Lanes 1 and 4 contain only a small amount of protein because of limited acrosomal exocytosis. (c-e) Sperm acrosomal status and the formation and detachment of acrosomal shrouds upon co-incubation were monitored by flow cytometry of live spermatozoa in which the acrosomes were labeled with fluorescently-conjugated lectin PNA. (c) Gating of detached acrosomal shrouds (red dots) from spermatozoa (green dots) in scatter diagrams from flow cytometry of sperm-ZPP fractions. Each dot represents one flow cytometric event, a shroud or a sperm cell (2,000 events/fraction). (d) Number of intact acrosomal shrouds, gated in visible light scatter, is increased by addition of proteasomal inhibitors (lower right panel) to coincubation, compared to control sperm-ZPP fraction (upper right), and sperm fractions prior to (upper left) and after capacitation (upper right), not exposed to ZPP. A degree of spontaneous acrosomal exocytosis is expected during capacitation. (e) Percentage of detached acrosomal shrouds in the coincubation fraction (i.e. ratio of red dot-events to all events in the scatter diagrams gated on acrosomal shrouds), was increased significantly (ANOVA; p<0.05) by the addition of proteasomal inhibitor cocktail for the duration of sperm-ZPP coincubation.

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)

Soluble ZPC protein binds to capacitated spermatozoa and triggers acrosomal exocytosis and separation of the acrosomal shrouds.(a). Immunofluorescence of sperm receptor protein ZPC (red) immediately after ZPP-sperm mixing (left), after 2 h of incubation (center; note the detached acrosomal shrouds-arrowheads) and after 2 h incubation with proteasomal inhibitors (right; arrows point to sperm with attached shrouds). (b). Addition of proteasomal inhibitors to sperm-ZPP coincubation limited the release of acrosomal proteasomes. Supernatant fractions (left panel) were collected from 10,000 sperm prior to mixing with ZPP (lane 1), after 2 h of coincubation (lane 2; appropriate vehicles were present), after 2 h co-incubation with proteasomal inhibitors added (lane 3) and immediately after sperm-ZPP mixing. Proteasomes were detected with a monoclonal antibody against alpha-type 20S proteasomal core subunits. The right panel shows the corresponding residual PAGE gel after protein transfer, confirming comparable protein loads between vehicle and inhibitor lanes (lanes 2&3). Lanes 1 and 4 contain only a small amount of protein because of limited acrosomal exocytosis. (c-e) Sperm acrosomal status and the formation and detachment of acrosomal shrouds upon co-incubation were monitored by flow cytometry of live spermatozoa in which the acrosomes were labeled with fluorescently-conjugated lectin PNA. (c) Gating of detached acrosomal shrouds (red dots) from spermatozoa (green dots) in scatter diagrams from flow cytometry of sperm-ZPP fractions. Each dot represents one flow cytometric event, a shroud or a sperm cell (2,000 events/fraction). (d) Number of intact acrosomal shrouds, gated in visible light scatter, is increased by addition of proteasomal inhibitors (lower right panel) to coincubation, compared to control sperm-ZPP fraction (upper right), and sperm fractions prior to (upper left) and after capacitation (upper right), not exposed to ZPP. A degree of spontaneous acrosomal exocytosis is expected during capacitation. (e) Percentage of detached acrosomal shrouds in the coincubation fraction (i.e. ratio of red dot-events to all events in the scatter diagrams gated on acrosomal shrouds), was increased significantly (ANOVA; p<0.05) by the addition of proteasomal inhibitor cocktail for the duration of sperm-ZPP coincubation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017256-g002: Soluble ZPC protein binds to capacitated spermatozoa and triggers acrosomal exocytosis and separation of the acrosomal shrouds.(a). Immunofluorescence of sperm receptor protein ZPC (red) immediately after ZPP-sperm mixing (left), after 2 h of incubation (center; note the detached acrosomal shrouds-arrowheads) and after 2 h incubation with proteasomal inhibitors (right; arrows point to sperm with attached shrouds). (b). Addition of proteasomal inhibitors to sperm-ZPP coincubation limited the release of acrosomal proteasomes. Supernatant fractions (left panel) were collected from 10,000 sperm prior to mixing with ZPP (lane 1), after 2 h of coincubation (lane 2; appropriate vehicles were present), after 2 h co-incubation with proteasomal inhibitors added (lane 3) and immediately after sperm-ZPP mixing. Proteasomes were detected with a monoclonal antibody against alpha-type 20S proteasomal core subunits. The right panel shows the corresponding residual PAGE gel after protein transfer, confirming comparable protein loads between vehicle and inhibitor lanes (lanes 2&3). Lanes 1 and 4 contain only a small amount of protein because of limited acrosomal exocytosis. (c-e) Sperm acrosomal status and the formation and detachment of acrosomal shrouds upon co-incubation were monitored by flow cytometry of live spermatozoa in which the acrosomes were labeled with fluorescently-conjugated lectin PNA. (c) Gating of detached acrosomal shrouds (red dots) from spermatozoa (green dots) in scatter diagrams from flow cytometry of sperm-ZPP fractions. Each dot represents one flow cytometric event, a shroud or a sperm cell (2,000 events/fraction). (d) Number of intact acrosomal shrouds, gated in visible light scatter, is increased by addition of proteasomal inhibitors (lower right panel) to coincubation, compared to control sperm-ZPP fraction (upper right), and sperm fractions prior to (upper left) and after capacitation (upper right), not exposed to ZPP. A degree of spontaneous acrosomal exocytosis is expected during capacitation. (e) Percentage of detached acrosomal shrouds in the coincubation fraction (i.e. ratio of red dot-events to all events in the scatter diagrams gated on acrosomal shrouds), was increased significantly (ANOVA; p<0.05) by the addition of proteasomal inhibitor cocktail for the duration of sperm-ZPP coincubation.
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