Investig Clin Urol.  2019 Jul;60(4):303-311. 10.4111/icu.2019.60.4.303.

Human spermatozoa anti-proprotein convertase subtilisin/kexin type 4 synthesis using New Zealand rabbit for novel immunocontraception in males

• Dahril
• Aulanni'am
• Keumala V
• Mustafa A

• KMID: 2450548
• DOI: http://doi.org/10.4111/icu.2019.60.4.303

Abstract

PURPOSE
Proprotein convertase subtilisin/kexin type 4 (PCSK4, a 54-kDa protease) is expressed in the plasma membrane of the acrosome in human spermatozoa. It plays a critical role in penetrating the zona pellucida. Synthesis of human anti-PCSK4 might be important for novel male immunocontraception.
MATERIALS AND METHODS
We used semen from adult males as the source of antigen (acrosomal PCSK4). Isolation and antigen characterization were done by immunohistochemistry followed by electrophoresis. Purification of PCSK4 was done by the electroelution method, followed by immunization with an animal model (Oryctolagus cuniculus). Antibody was collected, purified, and tested by using Western blot and dot blot. Antibody in vitro potential testing was performed on human spermatozoa by laser scanning microscopy with rhodamine stain under a light microscope and on rat spermatozoa.
RESULTS
Human anti-PCSK4 bound with PCSK4 on the head of human spermatozoa and could interfere with rat spermatozoa activity to penetrate the oocyte.
CONCLUSIONS
The result of this study can be used as a basis to develop new immunocontraceptives targeted towards males. This study shows that antibodies from induction of PCSK4 from spermatozoa may hinder fertilization.

Keyword

Antibodies; Contraception, immunologic; Proprotein convertases

MeSH Terms

Acrosome
Adult
Animals
Antibodies
Blotting, Western
Cell Membrane
Contraception, Immunologic*
Electrophoresis
Fertilization
Head
Humans*
Immunization
Immunohistochemistry
In Vitro Techniques
Male*
Methods
Microscopy, Confocal
Models, Animal
New Zealand*
Oocytes
Proprotein Convertases
Rabbits*
Rats
Rhodamines
Semen
Spermatozoa*
Zona Pellucida
Antibodies
Proprotein Convertases
Rhodamines

Figure

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  Fig. 1 Isolation of 54-kDa protein in human spermatozoa membrane. PBS, phosphate-buffered saline; PBS-T, PBS-Tween 20; PMSF, phenylmethylsulfonyl fluoride.

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  Fig. 2 Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) electrophoresis of human spermatozoa protein membrane. RSB, reducing sample buffer.

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  Fig. 3 Purification of proteins by electroelution method.

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  Fig. 4 Purification of serum antibody. SAS, supercritical antisolvent.

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  Fig. 5 Step by step sequence for performing specificity test using dot blotting. PBS, phosphate-buffered saline; IgG, immunoglobulin G; FFB, final formulation buffer; AP, alkaline phosphatase.

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  Fig. 6 Step by step sequence for specificity test using Western blot method. SDS-PAGE, Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis; NC, nitrocellulose; PBS, phosphate-buffered saline; PBS-T, PBS-Tween 20; AP, alkaline phosphatase; FFB, final formulation buffer.

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  Fig. 7 Proprotein convertase subtilisin/kexin type 4 (PCSK4) expression on the surface of spermatozoa plasma membrane. Arrows: PCSK4 expression on the acrosome (immunohistochemistry method using DAB chromogen, counterstain using methyl green, ×100).

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  Fig. 8 Specificity test of anti-proprotein convertase subtilisin/kexin type 4 (PCSK4) to PCSK4 using Western blot and dot blot technique.

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  Fig. 9 Ligand binding receptor (proprotein convertase subtilisin/kexin type 4 [PCSK4] with human anti-PCSK4) with confocal laser scanning microscope (CLSM) examination. Green color (yellow circles) is PCSK4 bound with anti-PCSK4 on the head of human spermatozoa. Blue circles highlight the normal appearance of the spermatozoa head.

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  Fig. 10 Spermatozoa receptor and oocyte binding inhibition. (A) Rat spermatozoa without anti-proprotein convertase subtilisin/kexin type 4 (PCSK4) immunization can move and bind to oocyte extract on the object glass. (B) Rat spermatozoa with anti-PCSK4 immunization move away from the oocyte extract. Arrows: rat oocyte extract on the object glass. Circles explain that the spermatozoa move away from the oocyte.

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