제   목 Acrosomal localization of LYZL6 on spermatozoa and its role in fertilization
등록일 2015/12/18 조회수 429 첨부파일
2015 PSRM 10th Biennial Meeting
2015 KSRM 69th Annual Meeting

Sojung Kwon, Hyun-Chul Shin, Dong-Won Seol, Jin Il Lee, Dong Hyuk Shin, and Dong Ryul Lee

Introduction
Lysozyme-like proteins (LYZLs) belong the class of chicken-type (c-type) lysozymes and are not well characterized in many species including mice and human. The c-type lysozyme genes (Lyzls) are all highly expressed in the testis and epididymis. In four c-type lysozyme genes (Lyzl1, Lyzl3/Spaca3, Lyzl4 and Lyzl6), the function of Lyzl1 and Lyzl6 still has not been clarified. Regarding to the biological function of Lyzl6, it has recently been reported that the presence of LYZL6 protein in primary spermatocytes and round spermatids of the testis and on the post-acrosomal area and midpiece of mature epididymal spermatozoa in mice and the antibacterial activity of recombinant LYZL6 protein.
  Although the expression pattern of LYZL6 in male germ cells was identified, its role in reproduction such as sperm-egg binding or fertilization like Spaca3 and Lyzl4 has not been fully examined.
  In this study, we analyzed the expression of Lyzl6 in both mice and human and possible contribution of LYZL6 in fertilization.

Material & method
Antibody
We prepared custom-raised rabbit polyclonal anti-LYZL6 sera used the     c-terminus of mouse LYZL6 protein as an epitope.
RT-PCR
Amplification of housekeeping 18S ribosomal protein gene was performed as internal control.
In situ hybridization
Paraffin sections of mouse or human testes were probed with cRNA antisense or sense probes for control purpose.
Immunofluorescence staining
Paraffin sections of mouse testes were used. Peanut agglutinin (PNA) was used as an acrosome marker.
Acrosome reaction
Artificial acrosome reaction of sperm was induced by using calcium ionophore.
In vitro fertilization (IVF)
C57BL/6J mice were used in experiment. IVF was performed in the presence of anti-LYZL6 sera or pre-immune sera.

Summary
  Consistent with the previous reports, Lyzl6 expressed in mice and human testis. In mice, the unique expression pattern of LYZL6 in the testis was detected from 3 weeks and colocalized with PNA, a marker for the acrosomal region, on the spermatids and spermatozoa. After the acrosome reaction by using calcium ionophore, the fluorescence signal of LYZL6 on the spermatozoa was disappeared. Treatment of anti-LYZL6 sera during in vitro fertilization resulted in a significant inhibition of fertilization (P ≤ 0.05).

Conclusion
Collectively, these results suggest that LYZL6 has a reproductive role as an acrosomal component on the spermatozoa during fertilization.