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Identification of phospholipase c zeta in mouse testes by immunocytochemistry
- 언어ENG
- URLhttps://db.koreascholar.com/Article/Detail/355156
Mature mammalian oocytes are ovulated at the metaphase II stage of meiosis and complete the cell cycle by fertilization with sperm. During fertilization sperm release an egg activation protein, pho-spholipase C zeta (PLCZ) into oocyte cytoplasm, PLCZ hydrolyze PIP2 into IP3 and DAG. The elevation of IP3 concentration induces Ca2+ release from endoplasmic reticulum (ER) by binding to the IP3 receptor (IP3R) on the membrane of ER. Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca2+]i oscillations or oocyte activation. Purified recombinant human PLCZ1 (hPLCZ1) protein evaluated its [Ca2+]i oscillation activity in mouse and human oocytes. Here we investigated that produced mouse PLCZ-specific antibodyrecognized the PLCZ protein in mouse testes. PLCZ antibody was raised in rabbits against 19-mer sequence at the C-terminus (MENKWFLSMVRDDFKGGKI) of mouse PLCZ protein. Sperm were fixed in 3.7% paraformaldehyde followed by permeabilization. Sperm were incubated in 5% normal goat serum (NGS) and then incubated overnight with anti-mouse PLCZ. Peanut agglutinin (PNA)-lectin was used for detection of the acrosome. Mouse testes from 6~8 weeks old ICR mouse were fixed in 10% formalinand serial sectioned at 5~8um. Testes tissues were immunostained with anti PLCZ antibody and peanut agglutinin(PNA) for acrosome staining. Produced anti mouse PLCZ antibody recognized 74 kDa protein in western and PLCZ is localized to the post-acrosomal region of mouse sperm and to the equatorial region of bull sperm. Mouse PLCZ protein wasdetected on spermatocytes, spermatid, but not on spermatogonia in seminiferous tubules. Some residual bodies on sperm neck and tail showed strong signal of PLCZ, but this staining was still present with antigenic peptide pretreatment to reduce non specific antibody reaction. Also this antibody reacted with the apical region (arrowheads) of principal cells, where secretory vesicles accumulate on the epididymal tissue. But antigenic peptide pretreatment did not remove this apical region staining. This study presents PLCZ protein is localized on the post-acrosomal region or equatorial region of mouse and bull sperm head. Also PLCZ protein in mouse testes expressed from spermatocytes to mature sperm on later stage of spermatogenesis.
