Ubiquitin-proteasome system participates in the de-aggregation of spermadhesins and DQH protein during boar sperm capacitation

in Reproduction
Correspondence should be addressed to M Zigo; Email: zigom@missouri.edu or michal_zigo_2000@yahoo.com

We studied the participation of the ubiquitin-proteasome system (UPS) in spermadhesin release during in vitro capacitation (IVC) of domestic boar spermatozoa. At ejaculation, boar spermatozoa acquire low molecular weight (8–16 kDa) seminal plasma proteins, predominantly spermadhesins, aggregated on the sperm surface. Due to their arrangement, such aggregates are relatively inaccessible to antibody labeling. As a result of de-aggregation and release of the outer layers of spermadhesins from the sperm surface during IVC, antibody labeling becomes feasible in the capacitated spermatozoa. In vivo, the capacitation-induced shedding of spermadhesins from the sperm surface is associated with the release of spermatozoa from the oviductal sperm reservoir. We took advantage of this property to perform image-based flow cytometry to study de-aggregation and shedding of boar spermadhesins (AQN, AWN, PSP protein families) and boar DQH (BSP1) sperm surface protein which induces higher fluorescent intensity in capacitated vs ejaculated spermatozoa. Addition of a proteasomal inhibitor (100 µM MG132) during IVC significantly reduced fluorescence intensity of all studied proteins (P < 0.05) compared to vehicle control IVC. Western blot detection of spermadhesins did not support their retention during IVC with proteasomal inhibition (P > 0.99) but showed the accumulation of DQH (P = 0.03) during IVC, compared to vehicle control IVC. Our results thus demonstrate that UPS participates in the de-aggregation of spermadhesins and DQH protein from the sperm surface during capacitation, with a possible involvement in sperm detachment from the oviductal sperm reservoir and/or sperm-zona pellucida interactions. The activity of sperm UPS modulates de-aggregation of boar spermadhesins and DQH sperm surface protein/binder of sperm1 (BSP1) during the sperm capacitation.

Downloadable materials

  • Supplementary Fig. 1: (A) Flow cytometric measurements of the outer acrosomal membrane (OAM) remodeling during in vitro capacitation under the proteasomal activity permissive/inhibiting conditions and with vehicle control. The 2 % formaldehyde fixed spermatozoa were labeled with lectin PNA (peanut agglutinin). 0.2 % (v/v) EtOH was used as a vehicle and 100 μM MG132 for proteasome inhibiting conditions. As described in our previous study (my UPS paper), four sperm populations were distinguished and gated. Population gating and comparisons confirmed that populations 1, and 3 differsignificantly (P< 0.05, marked red) when compared to vehicle control (0.2 % EtOH), depending on proteasomal inhibition (100 μM MG 132) during capacitation. (B) Bar graph representation of flow cytometric measurements of the outer acrosomal membrane (OAM) remodeling during in vitro capacitation with proteasomal modulation and vehicle control. Results are presented as mean ± SD of four independent replicates. Sperm treatments are compared within each group (population) only. For simplicity, eachbar within a population is statistically compared to all bars to its left, and the statistical significance with p-value < 0.05 is represented by a superscript above the corresponding bar.
  • Supplementary Fig. 2: Flow cytometric measurements of boar spermatozoa from Fig. 1, before and after capacitation under proteasomal activity permissive/inhibiting (100 μM MG132) conditions, including vehicle control (0.2% EtOH). Spermatozoa were labelled with an antibody against AQN-1 spermadhesin to screen for AQN-1 de-aggregation, and lectin PNA for outer acrosomal membrane integrity screening. A density scatted plot represents differnet, more precise, AQN-1 labeling of each sperm treatment: before and after capacitation with/without proteasomal inhibitors, including vehicle control. Each scatter plot represents 10,000 properly focused single cell flow cytometric events. The experiment was replicated five times with comparable results.
  • Supplementary Fig. 3: Flow cytometric measurements of boar spermatozoa from Fig. 2, before and after capacitation under proteasomal activity permissive/inhibiting (100 μM MG132) conditions, including vehicle control (0.2% EtOH). Spermatozoa were labelled with an antibody against AWN spermadhesin to screen for AWN de-aggregation, and lectin PNA for outer acrosomal membrane integrity screening. A density scatted plot represents differnet, more precise, AWN labeling of each sperm treatment: before and after capacitation with/without proteasomal inhibitors, including vehicle control. Each scatter plot represents 10,000 properly focused single cell flow cytometric events. The experiment was replicated five times with comparable results.
  • Supplementary Fig. 4: Flow cytometric measurements of boar spermatozoa from Fig. 3, before and after capacitation under proteasomal activity permissive/inhibiting (100 μM MG132) conditions, including vehicle control (0.2% EtOH). Spermatozoa were labelled with an antibody against PSP-I spermadhesin to screen for PSP-I de-aggregation, and lectin PNA for outer acrosomal membrane integrity screening. A density scatted plot represents differnet, more precise, PSP-I labeling of each sperm treatment: before and after capacitation with/without proteasomal inhibitors, including vehicle control. Each scatter plot represents 10,000 properly focused single cell flow cytometric events. The experiment was replicated four times with comparable results.
  • Supplementary Fig. 5: Flow cytometric measurements of boar spermatozoa from Fig. 4, before and after capacitation under proteasomal activity permissive/inhibiting (100 μM MG132) conditions, including vehicle control (0.2% EtOH). Spermatozoa were labelled with an antibody against PSP-II spermadhesin to screen for PSP-II de-aggregation, and lectin PNA for outer acrosomal membrane integrity screening. A density scatted plot represents differnet, more precise, PSP-II labeling of each sperm treatment: before and after capacitation with/without proteasomal inhibitors, including vehicle control. Each scatter plot represents 10,000 properly focused single cell flow cytometric events. The experiment was replicated four times with comparable results.
  • Supplementary Fig. 6: Flow cytometric measurements of boar spermatozoa from Fig. 5, before and after capacitation under proteasomal activity permissive/inhibiting (100 μM MG132) conditions, including vehicle control (0.2% EtOH). Spermatozoa were labelled with an antibody against DQH (pB1, fibronectin type II domain containing protein, Fn2) to screen for DQH de-aggregation, and lectin PNA for outer acrosomal membrane integrity screening. A density scatted plot represents differnet, more precise, DQH labeling of each sperm treatment: before and after capacitation with/without proteasomal inhibitors, including vehicle control. Each scatter plot represents 10,000 properly focused single cell flow cytometric events. The experiment was replicated four times with comparable results.
  • Supplementary Fig. 7: (A) WB detection of DQH in SDS protein extracts from ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting (100 μM MG132) conditions and vehicle control (0.2% EtOH). Membranes were co-incubated with antibodies against DQH and β- tubulin. PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane within each treatment. B-tubulin detection and CBB stained residual gel after electro-transfer were used for protein load normalization purposes. Proteins were resolved on 8-20% gradient gel under non-reducing conditions, and protein equivalent of 10 million spermatozoa was loaded per single lane. The experiment was replicated eight times with comparable results. (B) Quantification of DQH detection on Western blot quantification from Fig. 5C and Fig. 5 table. Results are presented as mean ± SD of eight independent replicates. For simplicity, each bar within one population is statistically compared to all bars to its left only and the statistical significance with p-value < 0.05 is represented by a superscript over a corresponding bar.

 

    Society for Reproduction and Fertility

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    (A) Flow cytometric measurements of AQN-1 de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of AQN-1 localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of AQN-1 spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of five and eight independent replicates for flow cytometry and Western blot, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.

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    (A) Flow cytometric measurements of AWN de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of AWN localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of AWN spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of five and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.

  • View in gallery

    (A) Flow cytometric measurements of PSP-I de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of PSP-I localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of PSP-I spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of four and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.

  • View in gallery

    (A) Flow cytometric measurements of PSP-II de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of PSP-II localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of PSP-II spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± SD of four and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.

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    (A) Flow cytometric measurements of DQH (pB1) de-aggregation during in vitro capacitation under proteasome permissive/inhibiting conditions (100 µM MG132) and vehicle control (0.2% EtOH), combined with epifluorescence imaging of DQH localization (B–B″). Every flow cytometric run represents 10,000 events. (C) Western blot detection of DQH spermadhesin in the ejaculated and capacitated spermatozoa under proteasome permissive/inhibiting conditions (100 μM MG132) including vehicle control, with densitometric analysis (lower insert) and β-tubulin for protein normalization purposes (upper insert); (C′) the PVDF membrane stained with CBB after chemiluminescence detection shows comparable protein loads per lane, (C″) residual gel after electrotransfer for protein normalization purposes. Upper and lower inserts in C were cropped from the identical membrane and only differ in exposure times. Proteins were extracted with Laemmli loading buffer supplemented with 50 mM DTT, resolved on a 4–20% gradient gel, and protein equivalent of 10 million spermatozoa was loaded per single lane. Results are presented as mean ± s.d. of four and eight independent replicates for flow cytometry and Western blotting, respectively. Differences in proteasomal inhibition with statistical significance (P < 0.05) are highlighted in red. Cauda epididymal spermatozoa were used as a negative control for both flow cytometric measurements and WB detection.

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