The roles of protein disulphide isomerase family A, member 3 (ERp57) and surface thiol/disulphide exchange in human spermatozoa-zona pellucida binding.
Are multimeric sperm plasma membrane protein complexes, ERp57 and sperm surface thiol content involved in human spermatozoa-zona pellucida (ZP) interaction?



ERp57 is a component of a multimeric spermatozoa-ZP receptor complex involved in regulation of human spermatozoa-ZP binding via up-regulation of sperm surface thiol content.



A spermatozoon acquires its fertilization capacity within the female reproductive tract by capacitation. Spermatozoa-ZP receptor is suggested to be a composite structure that is assembled into a functional complex during capacitation. Sperm surface thiol content is elevated during capacitation. ERp57 is a protein disulphide isomerase that modulates the thiol-disulphide status of proteins.



The binding ability and components of protein complexes in extracted membrane protein fractions of spermatozoa were studied. The roles of capacitation, thiol-disulphide reagent treatments and ERp57 on sperm functions and sperm surface thiol content were assessed.



Spermatozoa were obtained from semen samples from normozoospermic men. Human oocytes were obtained from an assisted reproduction programme. Blue native polyacrylamide gel electrophoresis, western ligand blotting and mass spectrometry were used to identify the components of solubilized ZP/ZP3-binding complexes. The localization and expression of sperm surface thiol and ERp57 were studied by immunostaining and sperm surface protein biotinylation followed by western blotting. Sperm functions were assessed by standard assays.



Several ZP-binding complexes were isolated from the cell membrane of capacitated spermatozoa. ERp57 was a component of one of these complexes. Capacitation significantly increased the sperm surface thiol content, acrosomal thiol distribution and ERp57 expression on sperm surface. Sperm surface thiol and ERp57 immunoreactivity were localized to the acrosomal region of spermatozoa, a region responsible for ZP-binding. Up-regulation of the surface thiol content or ERp57 surface expression in vitro stimulated ZP-binding capacity of human spermatozoa. Blocking of ERp57 function by specific antibody or inhibitors against ERp57 reduced the surface thiol content and ZP-binding capacity of human spermatozoa.



N/A.



The mechanisms by which up-regulation of surface thiol content stimulates spermatozoa-ZP binding have not been depicted.



Thiol-disulphide exchange is a crucial event in capacitation. ERp57 modulates the event and the subsequent fertilization process. Modulation of the surface thiol content of the spermatozoa of subfertile men may help to increase fertilization rate in assisted reproduction.



This work was supported by The Hong Kong Research Grant Council Grant HKU764611 and HKU764512M to P.C.N.C. The authors have no competing interests.
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.
Chi-Wai
CW
Wong
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Centre of Reproduction, Development and Growth, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Hong Kong SAR, P.R. China.
Kevin K W
KKW
Lam
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Centre of Reproduction, Development and Growth, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Hong Kong SAR, P.R. China.
Cheuk-Lun
CL
Lee
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Centre of Reproduction, Development and Growth, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Hong Kong SAR, P.R. China.
William S B
WSB
Yeung
The Sixth Affiliated Hospital of Sun Yat-Sen University, Guangzhou, P.R. China.
Wei E
WE
Zhao
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Centre of Reproduction, Development and Growth, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Hong Kong SAR, P.R. China.
Pak-Chung
PC
Ho
Center for Reproductive Medicine, The Third Affiliated Hospital, Sun Yat-Sen University, Guangzhou, P.R. China.
Jian-Ping
JP
Ou
Department of Obstetrics and Gynaecology, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Centre of Reproduction, Development and Growth, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong, Hong Kong SAR, China.|Shenzhen Key Laboratory of Fertility Regulation, Department of Obstetrics and Gynecology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, Hong Kong SAR, P.R. China.
Philip C N
PCN
Chiu
D000177:Acrosome / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000502:physiology*; D013075:Sperm Capacitation; D013084:Sperm-Ovum Interactions; D013094:Spermatozoa / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism*; D015854:Up-Regulation; D015044:Zona Pellucida / Q000378:metabolism
D013438:Sulfhydryl Compounds; D019704:Protein Disulfide-Isomerases; C085868:PDIA3 protein, human
ERp57; PDIA3; capacitation; fertilization; sperm function; thiol-disulphide exchange; zona pellucida