Characterisation of Lyzls in mice and antibacterial properties of human LYZL6

Abstract

C-type lysozyme genes (Lyzls) belong to the class of lysozymes and are highly expressed in the testis and epididymis. The members Lyzl4 and Spaca3 have been reported to play a role in sperm-egg binding and fertilisation in mice. However, the function of the remaining two mouse c-type lysozyme genes, Lyzl1 and Lyzl6, is still not clear. In the present study, we analysed the tissue expression and androgen-dependent expression of mouse c-type lysozyme genes and the possible contribution of human recombinant LYZL6 (rLYZL6) to immunity. The expression of Lyzls was detected by RT-PCR, Western blots, immunohistochemistry and immunofluorescence. The bacteriolytic activity of rLYZL6 was analysed by a colony-forming assay. In mice, the expression of Lyzl genes was mainly in the testis and epididymis in a developmentally regulated manner and androgen- or testicular factor-regulated manner. Immunodetection revealed 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. The rLYZL6 protein exhibited antibacterial activity. From the results, Lyzls may play a role in mitochondrial function of spermatozoa and LYZL6 may contribute to the innate immunity of the male genital tract.

Figure 1

Expression of Lyzls in mice. Tissue distribution of Lyzl1, 3, 4 and 6 in adult mice. (a) RT-PCR analysis was performed on total RNA isolated from Te (testis), Ep (epididymis), Sv (seminal vesicle), Vd (vas deferens), Ki (kidney), Br (brain), He (heart), Li (liver), Sp (spleen), St (stomach), Lu (lung), Mu (muscle), Bl (bladder), Je (jejunum) and Ad (adrenal). Developmental expression of Lyzls transcripts in the testis (b) and epididymis (c) of mice with postnatal age. RT-PCR for Lyzl1, 3, 4 and Lyzl6 was performed using RNA isolated from the testis and epididymis of 14- to 90-day-old mice (abscissa). Gapdh served as internal control. Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

Figure 2

Expression of Lyzls in mouse epididymides following castration and subsequent testosterone manipulation. (a) RT-PCR detection of adult mouse epididymal RNA from males bilaterally castrated for 1, 3, 5 and 7 days (C1, C3, C5, C7), sham-operated males for 1 and 7 days (S1, S7) and 1, 3, 5 and 7 days after a single injection of testosterone propionate applied to 7-day castrated mice (C7+1, C7+3, C7+5 and C7+7) and sham-operated mice (S7+1, S7+3, S7+7 and S7+7). Total RNA was pooled from three animals at each time-point in castrated animals (b) and shams (c). The relative expression of Lyzls mRNA (expression of Lyzls mRNA/Gapdh mRNA) (in different colours and symbols) in the epididymides was compared with the serum testosterone level during androgen manipulation (n=3). Gapdh, glyceraldehyde-3-phosphate dehydrogenase.

Figure 3

Expression of Lyzl6 gene and LYZL6 protein in the mouse testis and epididymis. (a) Total RNA isolated from the adult testis, caput, corpus and cauda was amplified by RT-PCR. (b) Western blot analysis of LYZL6 in the adult testis and epididymis. (c) Immunohistochemical localisation of LYZL6 in the adult mouse testis (c1), initial segment (c3), caput (c5), corpus (c7) and cauda (c9); c2, c4, c6, c8 and c10 are the corresponding negative controls. LYZL6 was mainly localized in the nuclei of primary spermatocytes (pri), round spermatids (rsd). The spermatogonia (spe) and elongated spermatids (esd) were all negative for LYZL6 staining. Basal (bas) and principal (prin) cells were positive in the epididymal sections. Scale bar=20 µm.

Figure 4

Immunofluorescence of LYZL6 on spermatozoa. Testicular spermatozoa (a); spermatozoa from the caput (b), corpus (c) and cauda (d) epididymidis, and vas deferens (e); phase contrast view of spermatozoa (f). Scale bars=10 µm.

Figure 5

(a) Purification of GST-LYZL6. Induction and purification of GST-LYZL6 in E. coli shown by Coomassie blue staining. Lane 1, molecular weight markers; lane 2, total protein of E. coli induced for 4 h; lane 3, total protein of E. coli induced 4 h with half concentration of the protein in lane 2. (b) Antibacterial activity of rLYZL6 by incubation of S.au ATCC25923 for 15–120 min at 37 °C with 12.5 µg ml⁻¹ (diamonds), 25 µg ml⁻¹ (circles), 50 µg ml⁻¹ (triangles) and 100 µg ml⁻¹ (squares) rLYZL6. To determine the number of CFUs, serial dilutions were plated and colonies were counted the following day. Data present means±s.e.m. of triplicate samples. CFU, colony-forming unit; GST, glutathione S-transferase; rLYZL6, recombinant LYZL6.