Molecular cloning and characterization of phospholipase C zeta in equine sperm and testis reveals species-specific differences in expression of catalytically active protein.
Abstract: Oocyte activation at fertilization is brought about by the testis-specific phospholipase C zeta (PLCZ), owing to its ability to induce oscillations in intracellular Ca(2+) concentration ([Ca(2+)](i)). Whereas this is a highly conserved mechanism among mammals, important species-specific differences in PLCZ sequence, activity, and expression have been reported. Thus, the objectives of this research were to clone and characterize the intracellular Ca(2+)-releasing activity and expression of equine PLCZ in sperm and testis. Molecular cloning of equine PLCZ yielded a 1914-bp sequence that translated into a protein of the appropriate size (~73 kDa), as detected with an anti-PLCZ-specific antibody. Microinjection of 1 μg/μl of equine PLCZ cRNA supported [Ca(2+)](i) oscillations in murine oocytes that were of a higher relative frequency than those generated by an equivalent concentration of murine Plcz cRNA. Immunofluorescence revealed expression of PLCZ over the acrosome, equatorial segment, and head-midpiece junction; unexpectedly, PLCZ also localized to the principal piece of the flagellum in all epididymal, uncapacitated, and capacitated sperm. Immunostaining over the acrosome was abrogated after induction of acrosomal exocytosis. Moreover, injection of either sperm heads or tails into mouse oocytes showed that PLCZ in both fractions is catalytically active. Immunohistochemistry on equine testis revealed expression as early as the round spermatid stage, and injection of these cells supported [Ca(2+)](i) oscillations in oocytes. In summary, we report that equine PLCZ displays higher intrinsic intracellular Ca(2+)-releasing activity than murine PLCZ and that catalytically active protein is expressed in round spermatids as well as the sperm flagellum, emphasizing important species-specific differences. Moreover, some of these results may suggest potential novel roles for PLCZ in sperm physiology.
Publication Date: 2011-03-09 PubMed ID: 21389344DOI: 10.1095/biolreprod.110.089466Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- Non-U.S. Gov't
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
The research aims to understand the specific functions of phospholipase C zeta (PLCZ), an enzyme found in testes and assists in the activation of oocytes during fertilization, in equine (horse) sperm and testes. Differences in activity, expression, and sequence of PLCZ were observed, indicating species-specific variations.
Molecular Cloning and Characterization of PLCZ
- The researchers carried out molecular cloning of the equine PLCZ, which resulted in a sequence of 1914 base pairs (bp). This sequence translated into a protein of approximately 73 kDa, fitting the expected size identified with a specific anti-PLCZ antibody.
Equine PLCZ Induces Higher Frequency Calcium Oscillations
- Injection of equine PLCZ-associated ribonucleic acid (RNA) into murine (mouse) oocytes induced oscillations in intracellular calcium concentration. These oscillations were of a higher frequency compared to those generated by equivalent amounts of murine PLCZ RNA.
Expression and Localization of PLCZ in Sperm and Testes
- Immunofluorescence studies showed that PLCZ was present in different parts of the sperm, including the acrosome, equatorial segment, and head-midpiece junction. Interestingly, it was also identified in the principal piece of the sperm’s flagellum.
- This expression was still evident in all stages of sperm maturity, from the sperm in the epididymis to both uncapacitated and capacitated ones.
- Following an acrosomal exocytosis, there was no PLCZ staining over the acrosome, suggesting a release or removal of PLCZ.
Introducing Sperm Heads or Tails into Mouse Oocytes Reveals Catalytically Active PLCZ
- The researchers injected either sperm heads or tails into mouse oocytes. The PLCZ present in both these fractions showed catalytic activity, suggesting its likely role during fertilization.
PLCZ Expression Starts Early in the Spermatogenesis Process
- Immunohistochemistry studies on equine testis showed that the expression of PLCZ starts as early as the round spermatid stage. Further, the researchers were able to show that introducing these early stage sperm cells into oocytes induced calcium oscillations.
Conclusion
- The research indicates that equine PLCZ exhibits stronger intracellular calcium-releasing activity compared to its murine counterpart. Furthermore, PLCZ is expressed in multiple stages of spermatogenesis and found in various parts of the mature sperm, suggesting species-specific differences and previously unidentified roles in sperm physiology.
Cite This Article
APA
Bedford-Guaus SJ, McPartlin LA, Xie J, Westmiller SL, Buffone MG, Roberson MS.
(2011).
Molecular cloning and characterization of phospholipase C zeta in equine sperm and testis reveals species-specific differences in expression of catalytically active protein.
Biol Reprod, 85(1), 78-88.
https://doi.org/10.1095/biolreprod.110.089466 Publication
Researcher Affiliations
- Departments of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York, USA. sjb55@cornell.edu
MeSH Terms
- Amino Acid Sequence
- Animals
- Calcium Signaling
- Cloning, Molecular
- Female
- Horses / metabolism
- Immunohistochemistry
- Male
- Molecular Sequence Data
- Oocytes / metabolism
- Phosphoinositide Phospholipase C / chemistry
- Phosphoinositide Phospholipase C / metabolism
- Sequence Homology, Amino Acid
- Species Specificity
- Spermatozoa / enzymology
- Testis / enzymology
Citations
This article has been cited 20 times.Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
