Human fertilization: Epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3

J. A. Maldera; M. Weigel Muñoz; M. Chirinos; D. Busso; F. G.E. Raffo; M. A. Battistone; J. A. Blaquier; F. Larrea; P. S. Cuasnicu

doi:10.1093/molehr/gat092

Human fertilization: Epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3

J. A. Maldera, M. Weigel Muñoz, M. Chirinos, D. Busso, F. G.E. Raffo, M. A. Battistone, J. A. Blaquier, F. Larrea, P. S. Cuasnicu^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

42 Scopus citations

Abstract

Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm- ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein- protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

Original language	English
Article number	gat092
Pages (from-to)	341-349
Number of pages	9
Journal	Molecular Human Reproduction
Volume	20
Issue number	4
DOIs	https://doi.org/10.1093/molehr/gat092
State	Published - Apr 2014
Externally published	Yes

Bibliographical note

Funding Information:
This study was partially supported by WHO RMG grant (H9/TSA/037), National Research Council of Argentina (CONICET) grant (PIP 2009-290), and National Agency for Scientific and Technological Promotion (ANPCyT) grant (PICT 2012-2023) to P.S.C., National Council of Science and Technology of Mexico (CONACyT) grant to F.L. and M.C. The Latin American Program for Research in Sexual and Reproductive Health (PLISSER) fellowship to M.C.

Keywords

Fertilization
HCRISP1
Oocyte
Sperm
Zona pellucida

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@article{dce306b6602c4b1f90e9d8ae878fa56e,

title = "Human fertilization: Epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3",

abstract = "Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm- ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein- protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.",

keywords = "Fertilization, HCRISP1, Oocyte, Sperm, Zona pellucida",

author = "Maldera, {J. A.} and Mu{\~n}oz, {M. Weigel} and M. Chirinos and D. Busso and Raffo, {F. G.E.} and Battistone, {M. A.} and Blaquier, {J. A.} and F. Larrea and Cuasnicu, {P. S.}",

note = "Funding Information: This study was partially supported by WHO RMG grant (H9/TSA/037), National Research Council of Argentina (CONICET) grant (PIP 2009-290), and National Agency for Scientific and Technological Promotion (ANPCyT) grant (PICT 2012-2023) to P.S.C., National Council of Science and Technology of Mexico (CONACyT) grant to F.L. and M.C. The Latin American Program for Research in Sexual and Reproductive Health (PLISSER) fellowship to M.C.",

year = "2014",

month = apr,

doi = "10.1093/molehr/gat092",

language = "English",

volume = "20",

pages = "341--349",

journal = "Molecular Human Reproduction",

issn = "1360-9947",

publisher = "Oxford University Press",

number = "4",

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TY - JOUR

T1 - Human fertilization

T2 - Epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3

AU - Maldera, J. A.

AU - Muñoz, M. Weigel

AU - Chirinos, M.

AU - Busso, D.

AU - Raffo, F. G.E.

AU - Battistone, M. A.

AU - Blaquier, J. A.

AU - Larrea, F.

AU - Cuasnicu, P. S.

N1 - Funding Information: This study was partially supported by WHO RMG grant (H9/TSA/037), National Research Council of Argentina (CONICET) grant (PIP 2009-290), and National Agency for Scientific and Technological Promotion (ANPCyT) grant (PICT 2012-2023) to P.S.C., National Council of Science and Technology of Mexico (CONACyT) grant to F.L. and M.C. The Latin American Program for Research in Sexual and Reproductive Health (PLISSER) fellowship to M.C.

PY - 2014/4

Y1 - 2014/4

N2 - Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm- ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein- protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

AB - Human epididymal CRISP1 (hCRISP1) associates with sperm during maturation and participates in gamete fusion through egg complementary sites. Its homology with both rodent epididymal CRISP1 and CRISP4 reported to participate in the previous stage of sperm binding to the zona pellucida (ZP), led us to further investigate the functional role of hCRISP1 by studying its involvement in human sperm- ZP interaction. Human hemizona (HZ) were inseminated with human capacitated sperm in the presence of either anti-hCRISP1 polyclonal antibody to inhibit sperm hCRISP1, or bacterially-expressed hCRISP1 (rec-hCRISP1) to block putative hCRISP1 binding sites in the ZP. Results revealed that both anti-hCRISP1 and rec-hCRISP1 produced a significant inhibition in the number of sperm bound per HZ compared with the corresponding controls. The finding that neither anti-hCRISP1 nor rec-hCRISP1 affected capacitation-associated events (i.e. sperm motility, protein tyrosine phosphorylation or acrosome reaction) supports a specific inhibition at the sperm-egg interaction level. Moreover, immunofluorescence experiments using human ZP-intact eggs revealed the presence of complementary sites for hCRISP1 in the ZP. To identify the ligand of hCRISP1 in the ZP, human recombinant proteins ZP2, ZP3 and ZP4 expressed in insect cells were co-incubated with hCRISP1 and protein- protein interaction was analyzed by ELISA. Results revealed that rec-hCRISP1 mainly interacted with ZP3 in a dose-dependent and saturable manner, supporting the specificity of this interaction. Altogether, these results indicate that hCRISP1 is a multifunctional protein involved not only in sperm-egg fusion but also in the previous stage of sperm-ZP binding through its specific interaction with human ZP3.

KW - Fertilization

KW - HCRISP1

KW - Oocyte

KW - Sperm

KW - Zona pellucida

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SN - 1360-9947

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JO - Molecular Human Reproduction

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M1 - gat092

ER -

Human fertilization: Epididymal hCRISP1 mediates sperm-zona pellucida binding through its interaction with ZP3

Abstract

Bibliographical note

Keywords

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