Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (7): 1445-1457.doi: 10.3864/j.issn.0578-1752.2022.07.015

• ANIMAL SCIENCE·VETERINARY SCIENCE·RESOURCE INSECT • Previous Articles     Next Articles

Expression and Localization of LCN5 in Ram Reproductive Organs and Spermatozoa

DONG FuCheng(),MA ShuLi,SHI JuanJuan,ZHANG JunMei,CUI Yan,REN YouShe,ZHANG ChunXiang()   

  1. College of Animal Science, Shanxi Agricultural University, Taigu 030801, Shanxi
  • Received:2021-05-14 Accepted:2021-09-14 Online:2022-04-01 Published:2022-04-18
  • Contact: ChunXiang ZHANG E-mail:dfc970314@163.com;zhchx66@126.com

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

Fig. 1

Different segments of the epididymis and the vas deferens"

Table 1

Kinematic parameters of different segments of the epididymis, vas deferens and ejaculated spermatozoa"

组别
Group
A percent
(%)
C percent
(%)
D percent
(%)
VAP
(μm·s-1)
VSL
(μm·s-1)
VCL
(μm·s-1)
ALH
(μm)
BCF
(Hz)
WOB
(%)
LIN
(%)
STR
(%)
ED 0.86±
0.27d
8.24±
1.94c
90.91±
5.92a
14.05±
1.18e
5.97±
0.46e
25.27±
3.98c
1.04±
0.23c
0.97±
0.36d
46.71±
9.63e
21.64±
5.52d
40.80±
6.57c
E1 0.97±
0.45d
18.14±
2.78a
79.22±
9.98b
14.19±
2.6e
7.36±
1.04de
29.27±
4.85c
1.09±
0.36c
1.57±
0.59cd
59.07±
9.03bcde
33.25±
5.23bcd
54.14±
7.63b
E2 1.52±
0.63d
11.64±
3.1abc
86.88±
12.92ab
17.64±
2.86de
9.53±
1.27de
33.79±
3.82bc
2.08±
0.70b
2.56±
0.78bcd
57.58±
7.54cde
33.68±
5.95bcd
55.74±
6.86bcd
E3 2.12±
0.44d
10.27±
1.50abc
87.60±
4.69ab
19.33±
2.26de
10.10±
2.64de
40.31±
4.48bc
1.59±
0.46bc
2.32±
0.75bcd
51.61±
9.59d
34.12±
6.13bcd
53.40±
4.88b
E4 1.87±
0.32d
18.34±
3.13a
79.78±
7.06b
18.93±
2.70de
13.19±
2.50de
30.06±
8.9c
1.68±
0.54bc
3.19±
1.25bc
56.30±
7.18de
43.49±
8.54abc
62.53±
9.59ab
E5 6.22±
1.65d
10.01±
2.62abc
83.76±
7.38ab
26.13±
4.75d
15.60±
1.85d
46.89±
3.6b
2.09±
0.29b
4.58±
2.48b
55.27±
9.40de
32.64±
5.20cd
56.41±
5.91b
E6 64.55±
9.39b
17.72±
3.47ab
14.72±
5.98d
60.55±
8.33b
46.13±
6.46b
99.62±
9.86a
3.83±
0.26a
8.48±
1.10a
62.81±
6.18abc
45.65±
5.39a
72.26±
5.42a
E7 67.13±
3.10b
12.38±
1.99abc
20.48±
5.61d
72.18±
4.97a
54.16±
6.63ab
101.8±
9.86a
3.58±
0.19a
8.40±
0.98a
69.65±
7.42ab
52.03±
6.21a
74.28±
5.42a
VS 43.41±
7.99c
13.50±
2.40 abc
43.09±
9.15c
46.27±
3.76c
29.58±
5.20c
89.42±
11.67a
3.76±
0.54a
9.80±
1.65a
51.60±
5.03d
32.52±
5.16cd
62.27±
7.05ab
ES 76.79±
4.88a
8.60±
1.5bc
14.88±
5.35d
74.24±
9.91a
55.27±
7.38a
100.6±
15.37a
3.77±
0.35a
8.83±
1.06a
73.25±
6.07a
54.52±
6.44a
74.29±
4.18a

Fig. 2

Expression of LCN5 protein in the ram testis, different segments of the epididymis and Vas deferens A: LCN5 protein and β-actin protein; B: Quantification of LCN5 protein"

Fig. 3

Immunohistochemistry of LCN5 in the ram testes, different segments of the epididymis, and the vas deferens -1: positive field(10×); -2: positive field (40×); -3: negative field (40×); Scale bar =10 μm. Intertubular tissue (IT), lumen (Lu), blood vessels (Bv), principal cells (Pc), basal cells (Bc), clear cells (Cc), smooth muscle (SM) cells, spermatozoa (Sp), stereocilia (Sc)"

Fig. 4

Immuno fluorescence of spermatozoa LCN5 in the ram testes, different segments of the epididymis, and the vas deferens -1: Anti-LCN5 staining (40×); -2: DAPI staining (40×); -3: merge field (40×); -4: negative merge field (40×). Arrows indicate a positive signal at the acrosome cap of the sperm, and asterisks indicate a positive signal at the midpiece of the sperm. Scale bar = 10 μm"

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