Molecular cloning and characterization of phospholipase C zeta in equine sperm and testis reveals species-specific differences in expression of catalytically active protein.

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.