1. PCSK4-null sperm display enhanced protein tyrosine phosphorylation and ADAM2 proteolytic processing during in vitro capacitation 
Charles Gyamera-Acheampong, M.Sc., Julian Vasilescu, M.Sc., Daniel Figeys, Ph.D., Majambu Mbikay, Ph.D. 
Fertility and Sterility 
Volume 93, Issue 4, Pages (March 2010)
DOI: /j.fertnstert
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Lack of PCSK4 leads to hyper–tyrosine phosphorylation in PCSK4-null sperm. Percoll gradient centrifugation (PGC)–washed sperm from both PCSK4-null and wild-type (WT) mice were capacitated for 0–3 hours. Aliquots were taken every 30 minutes and immunoblotted for tyrosine phosphoproteins (Yp-proteins) using antiphosphotyrosine monoclonal antibody (mAb) 4G10. KO = PCSK4-null.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Effect of HCO3−, Ca2+, and albumin on observed hyper–tyrosine phosphorylation in PCSK4-null sperm. The PGC-washed sperm from both PCSK4-null and WT mice were capacitated for 0, 30, and 90 minutes in (A) complete capacitation medium, (B) capacitation medium devoid of HCO3−, (C) capacitation medium devoid of Ca2+, and (D) capacitation medium devoid of albumin; sperm aliquots were immunoblotted for sperm proteins phosphorylated at tyrosine residues using mAb 4G10. In all cases, α-tubulin was used as housekeeping protein. (Normalization for tubulin would reduce the intensity of phosphorylated bands, but the differences would still be observed). Abbreviations as in Figure 1.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Effect of H89 on observed hyper–tyrosine phosphorylation in PCSK4-null sperm. (A) PGC-washed sperm from both PCSK4-null and WT mice were capacitated for 0, 30, and 90 minutes in complete capacitation medium supplemented with H89 or dimethyl sulfoxide (DMSO; control). Sperm aliquots were immunoblotted for sperm proteins phosphorylated at tyrosine residues using mAb 4G10. In all cases, α-tubulin was used as internal control. (B) Total sperm proteins from WT and PCSK4-null mice were separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for AKAP4. Other abbreviations as in Figure 1.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Processing of proADAM2 (top) and proADAM3 (bottom) in mouse testis/sperm. (A) Testes from PCSK4-null and WT mice were homogenized in radioimmunoprecipitation assay buffer containing complete protease inhibitor cocktail and sonicated. Homogenates were cleared by centrifugation, and the supernatants separated on SDS-PAGE and immunoblotted for ADAM2 and ADAM3. (B) PGC-washed sperm from PCSK4-null and WT mice were capacitated for 0–2 hours and aliquots taken very 30 minutes. Samples were separated on SDS-PAGE and immunoblotted for ADAM2 and ADAM3. Abbreviations as in Figures 1 and 3.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

6. Figure 5
Effect of cholesterol efflux on the gradual conversion of ∼46-kDa form of ADAM2 to ∼27-kDa form. The PGC-washed sperm from PCSK4-null and WT mice were capacitated for 90 minutes in different capacitation media: uncapacitated sperm samples (lanes 1 and 2); medium containing albumin (lanes 3 and 4); medium containing methyl-β-cyclodextrin (MBCD) instead of albumin (lanes 5 and 6); medium with neither albumin nor MBCD (lanes 7 and 8); medium containing albumin and supplemented with ChS (lanes 9 & 10); medium containing MBCD instead of albumin and supplemented with cholesterol sulfate (ChS) (lanes 11 and 12); medium supplemented with ChS without albumin or MBCD (lanes 13 and 14). Samples were separated on SDS-PAGE and immunoblotted for ADAM2. Other abbreviations as Figures 1 and 3.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

7. Figure 6
Effect of general PCSK inhibitors on sperm proADAM2 processing. The PGC washed sperm from PCSK4-WT mouse was capacitated in complete medium for 0 minute (lane 1), 90 minutes without any chloromethylketone (lane 2), 90 minutes with Dec-RVRK-CMK (lane 3), and 90 minutes with Dec-RVKR-CMK (lane 4). Sperm samples were separated on tricine SDS-PAGE and immunoblotted for ADAM2. Abbreviations as in Figures 1 and 3.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

8. Figure 7
Amino acid sequence of preproADAM2-V5. The amino-terminal signal peptide is given in green, the prodomain in blue, and the carboxyl-terminal V5 sequence in purple. Putative PCSK4 cleavage sites are shown in red, the disintegrin domain in underscored boldface; the transmembrane domain in underscored green. The integrin-binding motif QDECD is highlighted in blue, and asparagine residues (N) susceptible to glycosylation in yellow.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions

9. Figure 8
Processing of mouse proADAM2 by PCSK4. (A) QBI293A cells were transfected with a pIRES vector for the expression of mouse proADAM2-V5 alone (lane 1) or together with a pCIneo vector for expression of FL-rPCSK4-V5 in differing amounts (PCSK4, lanes 2–5). The final amount of plasmid was kept constant by adding a pCIneo empty vector. After 36 hours, cell lysates were subjected to immunoblotting (IB) with mAb V5. The blot was stripped and reprobed with mAb α-tubulin to serve as the internal control for equal loading. The processed intermediate, ∼66-kDa form, increases as the amounts of PCSK4 decrease. (B) QBI293A cells were transfected with a pIRES vector for expression of mouse proADAM2-V5 alone (lane 1) or together with a pCIneo vector for expression of FL-rPCSK4-V5 in differing amounts (PCSK4, lanes 2–5). The final amount of plasmid was kept constant by adding a pCIneo empty vector. After 36 hours, cell lysates were subjected to immunoprecipitation (IP) with mAb V5, and precipitates were analyzed by IB with the same antibody. Possible processing intermediates increase as the amounts of FL-rPCSK4-V5 decrease.
Fertility and Sterility  , DOI: ( /j.fertnstert )
Copyright © 2010 American Society for Reproductive Medicine Terms and Conditions