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Figure 1. Acrosome-borne 26S proteasomes degrade sperm receptor protein ZPC, solubilized from mature oocytes' zonae.(a) Diagram of the experimental setup in which the ZP-proteins (ZPP), solubilized by non-degrading/non-reducing methods from 100 meiotically mature, fertilization-competent porcine oocytes, were coincubated for 2 h with 10,000 capacitated boar spermatozoa. Each lane in the Western blots shown in panels b & c represent this number of gametes/proteins. The ZPP binding induced acrosomal exocytosis, enabling the separation of ZPC and sperm proteasome-containing acrosomal shrouds from the exocytosed spermatozoa. (b) Western blotting of ZPC in these co-incubated fractions detected a low mass degradation product (âDeg.â bracket), which was present in the coincubated fraction after 2 h (âVeh.â lane) or after 2 h of coincubation in the presence of a proteasomal inhibitor cocktail composed of epoxomicin, clasto-lactacystin-beta lactone (CLβL) and MG132 (âInh.â lane), but not detectable in the ZPP fraction prior to coincubation (âNo Inc.â lane). Densitometry revealed a statistically significant reduction (73% over the vehicle lane) in the density of the measured ZPC-degradation product (data from three replicates). (c) Accelerated degradation of ZPC was accomplished with the addition of ubiquitin-aldehyde (UBAL), a modified ubiquitin molecule that increases proteasomal proteolytic activity. Note a unique, low molecular mass band of <9 kDa that is prominent in the UBAL lane.
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Figure 2. 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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Figure 3. 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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Figure 4. 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 1â3) and 1 hr (lanes 4â7) 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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Figure 5. Porcine zona pellucida proteins are ubiquitinated prior to fertilization.(a) Zona pellucida fragments separated from minced porcine ovaries were solubilized (Left; Coomassie-stained PAGE gel-lane 2; lane 1â=âprotein markers), and subjected to affinity purification of ubiquitinated proteins using the recombinant UBA domain of ubiquitin-binding protein p62 (center; PAGE gel, lane 2). The protein band between 70â80 kDa was reactive to anti-ubiquitin antibodies (Right; Western blot). (b) This band was excised and identified by MALDI-TOF MS as porcine sperm receptor component ZPC (identified fragments are underlined). (c). Ubiquitinated proteins from solubilized zonae from preselected, morphologically normal porcine metaphase-II oocytes were affinity purified on p62 matrix (lane 2; PAGE) and showed immunoreactivity to anti-ubiquitin antibodies (lane 3; Western). (d) Soluble proteins isolated by p62 affinity-purification were subjected to Nanospray LC-MS/MS spectroscopy adjusted for Gly-Gly modification, a fingerprint of ubiquitinated internal Lys-residues. Gly-Gly modifications were observed on all three components of porcine ZP, including ZPA, ZPB and ZPC, and in the positive control, the K-48 linked multi-ubiquitin chains, but not in the unconjugated monoubiquitin.
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Figure 6. Patterns of zona pellucida deposition and ZPC-ubiquitin colocalization in porcine ocyte-cumulus complexes isolated from small antral follicles.(a, b) Accumulation of ZPC (gray) in ridges (arrowheads) adjacent to zona-adhering corona radiata cells (blue â=â nuclei stained with DAPI). (c-e) Colocalization of ZPC (red) and ubiquitin (green) in the zona pellucida (c, d) and in the cytoplasm of corona radiata cells (e); DNA was counterstained with DAPI (blue). Western blotting of ZPC protein in the isolated cumulus/corona cells from 60 oocyte cumulus complexes (lane 1), in 60 zona-free oocytes (lane 2) and in soluble zona proteins isolated from 60 oocytes (lane 3).
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Figure 7. Proteasomal inhibitors protect sperm acrosomal surface-associated proteins from degradation during ZPP-induced acrosomal exocytosis.Sperm-ZPP supernatants containing acrosomal shrouds and soluble/shroud bound zona proteins after 2 h of sperm-ZPP coincubation were separated on 1D PAGE gels. Bands that differed between vehicle control-fraction (left lane) and inhibitor fraction (right lane) were excised and subjected to LC-MS/MS identification. All of the identified proteins are known to be acrosomal components. ACE2 was the only protein diminished in the inhibitor-exposed fraction.
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Figure 8. Degradation of Intact Oocyte Zonae by Isolated Sperm Proteasomes.Maturing ova were incubated with purified sperm proteasomes for four hours with non-activated (a, b) or heat activated (c, d) sperm proteosomes, fertilized in vitro, and processed with anti-ZPC antibody (red), DNA stain DAPI (blue) and acrosomal shroud marker - lectin PNA-FITC (green). Note sperm detachment, and a striking abrasion and loosening of the zona in the active proteasome-treated ova (c,
d). Preincubation with active proteasomes coincides with a reduced rate of polyspermic fertilization after IVF (e, f). IVF experiment was repeated three times, with total oocyte numbers shown above each column in panel f. Control (g; left) and proteasome treated (g; right) ova were also processed with anti-ZPC antibody immediately at the end of the 4 h coincubation, causing a pattern of zona digestion and abrasion (right zona) similar to that seen in IVF ova pre-treated with active proteasomes.
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