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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

Proteolysis of ZPC in the presence of isolated sperm acrosomal proteasomes is similar to that in live sperm-ZPC coincubation-fractions.(a) Western blot of coincubated fractions with anti-ZPC antibody. After two-hours of ZPP-sperm proteasome co-incubation, a familiar degradation product appears (vehicle lane), which is reduced by the addition of proteasomal inhibitors (Inhibitor Cocktail lane), and amplified by the stimulation of proteasomal activity with ubiquitin aldehyde (UBAL lane). (b) Densitometry data from three replicates (lower panel) averaged 86% reduction (p<0.002) in the presence of proteasomal inhibitors, and a 23% acceleration (p<0.01) of proteasomal proteolysis with UBAL. (c) Time-lapse Western blotting of ZPC, revealing the progress of ZPC degradation during zona-protein coincubation with sperm proteasomes. No degradation products were observed in ZPP preparation incubated for 2 h without addition of isolated proteasome (last lane). (d) Thirty-minute time lapse of sperm-ZPC coincubation, revealing the formation of degradation product as early as 5 min. after ZPC-proteasome mixing. (e) Replicate of the two-hour time lapse experiment with isolated sperm proteasomes and solubilized zona proteins (ZPP), with/without addition of proteasomal inhibitor cocktail (MG132, CLBL & Epoxomicin), which eliminated the degradation of ZPC at 30 min (lanes 1–3) and 1 hr (lanes 4–7) after the onset of coincubation. Vehicles (DMSO, EtOH; lanes 2 &5) had no effect on degradation of ZPC; ZPP incubation for up to 1 hr without addition of proteasomes (lane 7) did not produce a detectable degradation product.
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pone-0017256-g004: Proteolysis of ZPC in the presence of isolated sperm acrosomal proteasomes is similar to that in live sperm-ZPC coincubation-fractions.(a) Western blot of coincubated fractions with anti-ZPC antibody. After two-hours of ZPP-sperm proteasome co-incubation, a familiar degradation product appears (vehicle lane), which is reduced by the addition of proteasomal inhibitors (Inhibitor Cocktail lane), and amplified by the stimulation of proteasomal activity with ubiquitin aldehyde (UBAL lane). (b) Densitometry data from three replicates (lower panel) averaged 86% reduction (p<0.002) in the presence of proteasomal inhibitors, and a 23% acceleration (p<0.01) of proteasomal proteolysis with UBAL. (c) Time-lapse Western blotting of ZPC, revealing the progress of ZPC degradation during zona-protein coincubation with sperm proteasomes. No degradation products were observed in ZPP preparation incubated for 2 h without addition of isolated proteasome (last lane). (d) Thirty-minute time lapse of sperm-ZPC coincubation, revealing the formation of degradation product as early as 5 min. after ZPC-proteasome mixing. (e) Replicate of the two-hour time lapse experiment with isolated sperm proteasomes and solubilized zona proteins (ZPP), with/without addition of proteasomal inhibitor cocktail (MG132, CLBL & Epoxomicin), which eliminated the degradation of ZPC at 30 min (lanes 1–3) and 1 hr (lanes 4–7) after the onset of coincubation. Vehicles (DMSO, EtOH; lanes 2 &5) had no effect on degradation of ZPC; ZPP incubation for up to 1 hr without addition of proteasomes (lane 7) did not produce a detectable degradation product.

Mentions: To ascertain that the observed ZPC proteolysis was specifically due to the activity of sperm proteasomes, purified proteasomes were isolated from boar spermatozoa (see proteasome characterization data, Fig. S2) and incubated with solubilized ZP-proteins. The reaction was supplied with energy in the form of ATP which is abundantly present and available to proteasomes in the intact boar spermatozoa [24], but has to be supplied externally for the sustenance of isolated proteasomes. The pattern of ZPC degradation by the sperm-derived proteasomes was very similar to that observed after ZPP co-incubation with whole spermatozoa (Fig. 4a). Also similar to whole sperm coincubation with ZP-proteins, proteasomal inhibitors reduced ZPC-degradation by isolated sperm proteasomes by 86%, compared to vehicle group (Fig. 4b). Furthermore, acceleration of proteasomal proteolysis with UBAL increased the density of the 12 kDa ZPC-degradation product by 23%, compared to proteasomes alone (Fig. 4b). A time-lapse Western blotting experiment revealed a progressive degradation pattern with a most prominent degradation product observed at 30 min. of coincubation (Fig. 4c). This degradation product was already visible after first 5 min. of coincubation (Fig. 4d), suggesting that ZPC degradation by sperm proteasomes occurs very rapidly. No degradation products were observed in ZPP preparations incubated for up to 2 h without the addition of isolated proteasome (Fig. 4c, d). Similar to two-hour incubation, the proteasomal inhibitor cocktail prevented the degradation of ZPC at 30 min and 1 hr after the onset of coincubation (Fig. 4e).


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)

Proteolysis of ZPC in the presence of isolated sperm acrosomal proteasomes is similar to that in live sperm-ZPC coincubation-fractions.(a) Western blot of coincubated fractions with anti-ZPC antibody. After two-hours of ZPP-sperm proteasome co-incubation, a familiar degradation product appears (vehicle lane), which is reduced by the addition of proteasomal inhibitors (Inhibitor Cocktail lane), and amplified by the stimulation of proteasomal activity with ubiquitin aldehyde (UBAL lane). (b) Densitometry data from three replicates (lower panel) averaged 86% reduction (p<0.002) in the presence of proteasomal inhibitors, and a 23% acceleration (p<0.01) of proteasomal proteolysis with UBAL. (c) Time-lapse Western blotting of ZPC, revealing the progress of ZPC degradation during zona-protein coincubation with sperm proteasomes. No degradation products were observed in ZPP preparation incubated for 2 h without addition of isolated proteasome (last lane). (d) Thirty-minute time lapse of sperm-ZPC coincubation, revealing the formation of degradation product as early as 5 min. after ZPC-proteasome mixing. (e) Replicate of the two-hour time lapse experiment with isolated sperm proteasomes and solubilized zona proteins (ZPP), with/without addition of proteasomal inhibitor cocktail (MG132, CLBL & Epoxomicin), which eliminated the degradation of ZPC at 30 min (lanes 1–3) and 1 hr (lanes 4–7) after the onset of coincubation. Vehicles (DMSO, EtOH; lanes 2 &5) had no effect on degradation of ZPC; ZPP incubation for up to 1 hr without addition of proteasomes (lane 7) did not produce a detectable degradation product.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0017256-g004: Proteolysis of ZPC in the presence of isolated sperm acrosomal proteasomes is similar to that in live sperm-ZPC coincubation-fractions.(a) Western blot of coincubated fractions with anti-ZPC antibody. After two-hours of ZPP-sperm proteasome co-incubation, a familiar degradation product appears (vehicle lane), which is reduced by the addition of proteasomal inhibitors (Inhibitor Cocktail lane), and amplified by the stimulation of proteasomal activity with ubiquitin aldehyde (UBAL lane). (b) Densitometry data from three replicates (lower panel) averaged 86% reduction (p<0.002) in the presence of proteasomal inhibitors, and a 23% acceleration (p<0.01) of proteasomal proteolysis with UBAL. (c) Time-lapse Western blotting of ZPC, revealing the progress of ZPC degradation during zona-protein coincubation with sperm proteasomes. No degradation products were observed in ZPP preparation incubated for 2 h without addition of isolated proteasome (last lane). (d) Thirty-minute time lapse of sperm-ZPC coincubation, revealing the formation of degradation product as early as 5 min. after ZPC-proteasome mixing. (e) Replicate of the two-hour time lapse experiment with isolated sperm proteasomes and solubilized zona proteins (ZPP), with/without addition of proteasomal inhibitor cocktail (MG132, CLBL & Epoxomicin), which eliminated the degradation of ZPC at 30 min (lanes 1–3) and 1 hr (lanes 4–7) after the onset of coincubation. Vehicles (DMSO, EtOH; lanes 2 &5) had no effect on degradation of ZPC; ZPP incubation for up to 1 hr without addition of proteasomes (lane 7) did not produce a detectable degradation product.
Mentions: To ascertain that the observed ZPC proteolysis was specifically due to the activity of sperm proteasomes, purified proteasomes were isolated from boar spermatozoa (see proteasome characterization data, Fig. S2) and incubated with solubilized ZP-proteins. The reaction was supplied with energy in the form of ATP which is abundantly present and available to proteasomes in the intact boar spermatozoa [24], but has to be supplied externally for the sustenance of isolated proteasomes. The pattern of ZPC degradation by the sperm-derived proteasomes was very similar to that observed after ZPP co-incubation with whole spermatozoa (Fig. 4a). Also similar to whole sperm coincubation with ZP-proteins, proteasomal inhibitors reduced ZPC-degradation by isolated sperm proteasomes by 86%, compared to vehicle group (Fig. 4b). Furthermore, acceleration of proteasomal proteolysis with UBAL increased the density of the 12 kDa ZPC-degradation product by 23%, compared to proteasomes alone (Fig. 4b). A time-lapse Western blotting experiment revealed a progressive degradation pattern with a most prominent degradation product observed at 30 min. of coincubation (Fig. 4c). This degradation product was already visible after first 5 min. of coincubation (Fig. 4d), suggesting that ZPC degradation by sperm proteasomes occurs very rapidly. No degradation products were observed in ZPP preparations incubated for up to 2 h without the addition of isolated proteasome (Fig. 4c, d). Similar to two-hour incubation, the proteasomal inhibitor cocktail prevented the degradation of ZPC at 30 min and 1 hr after the onset of coincubation (Fig. 4e).

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