关键词: LCN5, 附睾不同区段, 精子成熟, 精子贮存, 公绵羊

Abstract:

【Objective】The aim of this experiment was to investigate the expression pattern and localization of LCN5 in the testis of ram, the different segments of the epididymis and spermatozoa among them, providing clues to further explore the function of LCN5 in ram reproductive organs. 【Method】All the samples were from 6 healthy 9-month-old rams with similar weight (65.23±1.95 kg). Three rams were used to collect ejaculated sperm via artificial vagina while the others for collecting the testes, epididymis, and vas deferens as well as the regional spermatozoa within these male reproductive ducts using aseptic castration. The right testis, efferent ducts (ED), caput (E1-E2), corpus (E3-E5), cauda (E6-E7) and vas deferens (VS) samples were collected and placed in liquid nitrogen and 4% paraformaldehyde, respectively; the spermatozoa from ejaculated semen, the right testis, various segments of the epididymis and the vas deferens were separated and determined by the CASA system. Western Blot and immunohistochemistry were performed to detect the localization of LCN5 in the testis, the various segments of the epididymis, and vas deferens, respectively. Immunofluorescence was carried out to examine the dynamic localization of LCN5 in the spermatozoa from the testis, different segment of epididymis and vas deferens. 【Result】 (1) The CASA results showed that the proportion of grade A and C spermatozoa and the sperm kinematic parameters were highest, including VAP, VSL, VCL, ALH, BCF, WOB, LIN and STR from the ejaculated sperm and spermatozoa in caudal epididymis, compared with those from the other segments of male reproductive ducts. Except for that, there were the more grade A spermatozoa in VS, which showed better motility than others. However, the spermatozoa from testis were completely immotile. (2)The Western Blot results discovered that the highest expression of LCN5 was found in the E1 segment, next in segment E2 and E7, which were more than in corpus, testis and efferent ducts (P<0.05). The quantitative analysis showed the LCN5 expression levels in these tissues was ordered as E1>E2>E6>E3≈E5≈E4≈E7>VS>ED>T. (3) Immunohistochemistry found that there were slight positive signals of LCN5 in the testicular elongated spermatids, the principal and basal cells of the efferent ducts, while strong positive signals could be detected in the principal and basal cells, stereocilia of epididymal epithelium in the caput, corpus, and cauda. Granular signals of LCN5 were scattered around the sperm in the epididymal lumen. Besides, the weak LCN5 positive signals were found in the smooth muscle cells of the vas deferens. (4) Immunofluorescence results of LCN5 protein on the spermatozoa from different regions showed dynamic changes. Firstly, the local positive signals on the surface of sperm acrosome and midpiece of spermatozoa from testis and ED could be only found. With the spermatozoa transition from the testis to the epididymis, these signals began to expand and spread on the whole surface of sperm acrosome and the intensity of the signals on the sperm acrosome and midpiece reached the peak in the E1 segment. Secondly, the positive signals also existed in cytoplasmic droplets of the sperm flagellum. These signals also showed gradually loss with the removal of cytoplasmic droplets along the epidydimal ducts until E6 or E7. The expression pattern of LCN5 on the cauda and corpus on the sperm surface was similar to that of the caput. 【Conclusion】This research firstly revealed that LCN5 protein was regionally expressed in the ram reproductive system, highly expressed in the epididymal caput and gathered in the E6 segment. In the epididymal cauda, LCN5 was mainly expressed in the surface of acrosome on the spermatozoa from the caput, corpus, caudal of epididymis, as well as the cytoplasmic droplets of the sperm flagellum, which presented a dynamic distribution both on the surface of sperm and midpiece corresponding to the sperm quality and motility along the ram reproductive ducts, highlighting its potential function in sperm maturation. Taken together, LCN5 could play an essential role in spermatogenesis, maturation, storage, which provided the theoretical basis for its functional research and exploitation.

Key words: LCN5, different segments of the epididymis, sperm maturation, storage of sperm, ram