Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro.
Abstract: Oocyte activation is initiated when a fertilising spermatozoon delivers sperm-borne oocyte-activating factor(s) into the oocyte cytoplasm. Candidates for oocyte activation include two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (PAWP; also known as WBP2 N-terminal like (WBP2NL)). We localised PLCZ1 and WBP2NL/PAWP in stallion spermatozoa and investigated the PLCZ1 content and sperm parameters as well as cleavage after intracytoplasmic sperm injection (ICSI). PLCZ1 was identified as 71-kDa protein in the acrosomal and postacrosomal regions, midpiece and principal piece of the tail. Anti-WBP2NL antibody identified two WBP2NL bands (~28 and ~32kDa) in the postacrosomal region, midpiece and principal piece of the tail. PLCZ1 and WBP2NL expression was positively correlated (P=0.04) in sperm heads. Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labelled spermatozoa among stallions. PLCZ1 expression was significantly higher in viable than non-viable spermatozoa, and DNA fragmentation was negatively correlated with PLCZ1 expression and the percentage of positively labelled spermatozoa (P<0.05). The use of equine sperm populations considered to have high versus low PLCZ1 content resulted in significantly higher cleavage rates after ICSI of bovine and equine oocytes, supporting the importance of PLCZ1 for oocyte activation.
Publication Date: 2019-10-11 PubMed ID: 31597592DOI: 10.1071/RD19217Google Scholar: Lookup
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- Journal Article
Summary
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The research article explored the localization and importance of two proteins, phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2NL/PAWP), on stallion spermatozoa for oocyte activation during fertilization. The study used flow cytometry to quantify the PLCZ1 content in sperm and its association with success in vitro fertilization.
Protein Localization
- The researchers identified the presence and location of PLCZ1 and WBP2NL/PAWP protein on stallion spermatozoa.
- PLCZ1, identified as a 71-kDa protein, was found in the acrosomal and postacrosomal regions, midpiece, and principal piece of the sperm tail.
- Anti-WBP2NL antibodies detected two bands of WBP2NL (~28 and ~32kDa) in the postacrosomal region, midpiece, and principal piece of the sperm tail.
Protein Expression and Correlation
- Expression of PLCZ1 and WBP2NL in the sperm heads were found to be positively correlated.
- Flow cytometry evaluation of PLCZ1 revealed large variations in fluorescence intensity and the percentage of positively labeled spermatozoa among different stallions.
- Expression of PLCZ1 was significantly higher in viable spermatozoa compared to non-viable ones.
- A negative correlation was found between DNA fragmentation and both PLCZ1 expression and the percentage of positively labelled spermatozoa.
Connection to In Vitro Fertilization Success
- The use of equine sperm populations with high levels of PLCZ1 content led to significantly higher cleavage rates after intracytoplasmic sperm injection (ICSI) of bovine and equine oocytes.
- This supports the crucial role of PLCZ1 in oocyte activation, a key step in the process of fertilization.
Cite This Article
APA
Gonzalez-Castro RA, Amoroso-Sanches F, Stokes JE, Graham JK, Carnevale EM.
(2019).
Localisation of phospholipase Cζ1 (PLCZ1) and postacrosomal WW-binding protein (WBP2 N-terminal like) on equine spermatozoa and flow cytometry quantification of PLCZ1 and association with cleavage in vitro.
Reprod Fertil Dev, 31(12), 1778-1792.
https://doi.org/10.1071/RD19217 Publication
Researcher Affiliations
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, 3101 Rampart Rd, Fort Collins, Colorado, 80521, USA.
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, 3101 Rampart Rd, Fort Collins, Colorado, 80521, USA.
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, 3101 Rampart Rd, Fort Collins, Colorado, 80521, USA.
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, 3101 Rampart Rd, Fort Collins, Colorado, 80521, USA.
- Equine Reproduction Laboratory, Department of Biomedical Sciences, Colorado State University, 3101 Rampart Rd, Fort Collins, Colorado, 80521, USA; and Corresponding author. Email: elaine.carnevale@colostate.edu.
MeSH Terms
- Acrosome / metabolism
- Animals
- Cells, Cultured
- Cleavage Stage, Ovum / metabolism
- Embryo Culture Techniques / veterinary
- Embryo, Mammalian
- Female
- Flow Cytometry
- Horses / embryology
- Horses / metabolism
- Male
- Phosphoinositide Phospholipase C / analysis
- Phosphoinositide Phospholipase C / metabolism
- Seminal Plasma Proteins / metabolism
- Sperm Injections, Intracytoplasmic
- Spermatozoa / metabolism
- Tissue Distribution
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