CD18在附睾中的表达及其敲除对附睾功能的影响

Expression of CD18 in the epididymis and its impact on epididymal function after knockout

摘要

目的 明确小鼠附睾中CD18蛋白的表达和定位, 探讨敲除CD18对小鼠附睾功能的影响。方法 采用qRT-PCR和Western blotting检测CD18在成年C57BL/6雄性小鼠附睾和睾丸中的表达水平, 采用免疫组织化学和免疫荧光染色法检测小鼠附睾中CD18的表达。利用CRISPR-Cas9的核糖核蛋白构建CD18^-/-小鼠。计数成年CD18^-/-小鼠的前向运动精子、怀孕率和产仔数。采用HE染色和凋亡染色观察组织形态。结果 qRT-PCR和Western blotting显示, CD18在成年雄性小鼠睾丸中基本不表达, 但在附睾有表达; 免疫组织化学染色显示, CD18在成年小鼠附睾上皮细胞的细胞膜上有表达, 且主要集中在体部和尾部; CD18^-/-小鼠基本不育; HE染色和凋亡染色显示CD18^-/-小鼠附睾尾部精子数目大量减少且出现凋亡。结论 CD18在小鼠附睾上皮细胞的细胞膜上表达, 并且CD18^-/-小鼠附睾尾部精子减少且出现凋亡, 据此推测敲除内源性CD18表达可对附睾功能、雄性生育力产生重要影响。

Abstract

Objective To clarify the expression and localization of CD18 in the epididymis of mouse, and to investigate the influence of CD18 knockout on epididymal function of mouse. Methods qRT-PCR and Western blotting were applied to investigate the expression of CD18 in the epididymis and testis of adult C57BL/6 male mice. The expression of CD18 in the epididymis was assessed by immunohistochemistry and immunofluorescence staining. CD18 ^-/- mice were constructed by utilizing CRISPR-Cas9 ribonucleoprotein. The forward motile sperm, pregnancy rate, and litter size of adult CD18^-/- mice were counted. The tissue morphology was observed by HE staining and TUNEL staining. Results qRT-PCR and Western blotting analysis indicated that CD18 was basically not expressed in the testes of adult male mice, but was expressed in the epididymides. Immunohistochemical staining indicated that CD18 was expressed on the membrane of epididymal epithelium of adult mice, and was mainly concentrated in the corpus and cauda. CD18^-/- mice were basically infertile. HE staining and TUNEL staining showed significantly reduced sperm count and apoptosis in cauda epididymis of CD18^-/- mice. Conclusion CD18 is expressed on the cell membrane of epididymal epithelium of mouse. CD18^-/- mice show decreased sperm count and apoptosis in cauda epididymis. It is speculated that the elimination of endogenous CD18 expression may have an important effect on epididymal function and male fertility.

关键词

CD18 / 附睾上皮细胞 / 精子 / 小鼠

Key words

CD18 / epididymal epithelium / sperm / mouse

参考文献

[1] VINAY L, HESS R A, BELLEANN E C. Human efferent ductules and epididymis display unique cell lineages with motile and primary Cilia[J]. Andrology, 2025, 13(4): 894-910. DOI: 10.1111/andr.13732.

[2] BAADE T, MICHAELIS M, PRESTEL A, et al. A flexible loop in the paxillin LIM3 domain mediates its direct binding to integrin β subunits[J]. PLoS Biol, 2024, 22(9): e3002757. DOI: 10.1371/journal.pbio.3002757.

[3] WANG H L, PETERS T, SINDRILARU A, et al. Key role of macrophages in the pathogenesis of CD18 hypomorphic murine model of psoriasis[J]. J Invest Dermatol, 2009, 129(5): 1100-1114. DOI: 10.1038/jid.2009.43.

[4] MAIGUEL D, FARIDI M H, WEI C L, et al. Small molecule-mediated activation of the integrin CD11b/CD18 reduces inflammatory disease[J]. Sci Signal, 2011, 4(189): ra57. DOI: 10.1126/scisignal.2001811.

[5] LIU Y, MA S L, WANG X B, et al. The role of β2 integrin associated heparin-binding protein release in ARDS[J]. Life Sci, 2018, 203: 92-98. DOI: 10.1016/j.lfs.2018.04.029.

[6] WANG B M, LIM J H, KAJIKAWA T, et al. Macrophage β2-integrins regulate IL-22 by ILC3s and protect from lethal Citrobacter rodentium-induced colitis[J]. Cell Rep, 2019, 26(6): 1614-1626.e5. DOI: 10.1016/j.celrep.2019.01.054.

[7] HE Y N, LI J X, QU Y J, et al. Identification and analysis of potential immune-related biomarkers in endometriosis[J]. J Immunol Res, 2023, 2023: 2975581. DOI: 10.1155/2023/2975581.

[8] VOISIN A, SAEZ F, DREVET J R, et al. The epididymal immune balance: a key to preserving male fertility[J]. Asian J Androl, 2019, 21(6): 531-539. DOI: 10.4103/aja.aja_11_19.

[9] SHI X, ZHAO H, KANG Y F, et al. The role of mononuclear phagocytes in the testes and epididymis[J]. Int J Mol Sci, 2022, 24(1): 53. DOI: 10.3390/ijms24010053.

[10] RINALDI V D, DONNARD E, GELLATLY K, et al. An atlas of cell types in the mouse epididymis and vas deferens[J]. eLife, 2020, 9: e55474. DOI: 10.7554/eLife.55474.

[11] MENDELSOHN A C, SANMARCO L M, SPALLANZANI R G, et al. From initial segment to cauda: a regional characterization of mouse epididymal CD11c⁺ mononuclear phagocytes based on immune phenotype and function[J]. Am J Physiol Cell Physiol, 2020, 319(6): C997-C1010. DOI: 10.1152/ajpcell.00392.2020.

[12] TIAN F, LEI J, NI Y F, et al. Regulation of CD18 stability by SIGIRR-modulated ubiquitination: new insights into the relationship between innate immune response and acute lung injury[J]. FEBS J, 2023, 290(10): 2721-2743. DOI: 10.1111/febs.16708.

[13] BATTISTONE M A, MENDELSOHN A C, SPALLANZANI R G, et al. Region-specific transcriptomic and functional signatures of mononuclear phagocytes in the epididymis[J]. Mol Hum Reprod, 2020, 26(1): 14-29. DOI: 10.1093/molehr/gaz059.

[14] BRETON S, NAIR A V, BATTISTONE M A. Epithelial dynamics in the epididymis: role in the maturation, protection, and storage of spermatozoa[J]. Andrology, 2019, 7(5): 631-643. DOI: 10.1111/andr.12632.

[15] GUITON R, VOISIN A, HENRY-BERGER J, et al. Of vessels and cells: the spatial organization of the epididymal immune system[J]. Andrology, 2019, 7(5): 712-718. DOI: 10.1111/andr.12637.

[16] BHUSHAN S, SCHUPPE H C, FIJAK M, et al. Testicular infection: microorganisms, clinical implications and host-pathogen interaction[J]. J Reprod Immunol, 2009, 83(1/2): 164-167. DOI: 10.1016/j.jri.2009.07.007.

[17] CAO Z M, GARCIA M J, SKLAR L A, et al. A flow cytometry-based high-throughput technique for screening integrin-inhibitory drugs[J]. J Vis Exp, 2024(204): e64401. DOI: 10.3791/64401.

[18] GARCÍA-SALIDO A, SERRANO-GONZÁLEZ A, DE AZAGRA-GARDE A M, et al. Flow cytometry analysis of CD64, CD18, CD11a and CD11b in four children with Bordetella pertussis infection and admitted to critical care: new biomarkers?[J]. Med Intensiva, 2019, 43(7): 446-449. DOI: 10.1016/j.medin.2017.10.007.

[19] BEDNARCZYK M, STEGE H, GRABBE S, et al. β2 integrins-multi-functional leukocyte receptors in health and disease[J]. Int J Mol Sci, 2020, 21(4): 1402. DOI: 10.3390/ijms21041402.

[20] WIJAYARATHNA R, PASALIC A, NICOLAS N, et al. Region-specific immune responses to autoimmune epididymitis in the murine reproductive tract[J]. Cell Tissue Res, 2020, 381(2): 351-360. DOI: 10.1007/s00441-020-03215-8.

[21] WANG M, YANG Y L, CANSEVER D, et al. Two populations of self-maintaining monocyte-independent macrophages exist in adult epididymis and testis[J]. Proc Natl Acad Sci U S A, 2021, 118(1): e2013686117. DOI: 10.1073/pnas.2013686117.

[22] PLEUGER C, AI D D, HOPPE M L, et al. The regional distribution of resident immune cells shapes distinct immunological environments along the murine epididymis[J]. eLife, 2022, 11: e82193. DOI: 10.7554/eLife.82193.

[23] DA SILVA N, CORTEZ-RETAMOZO V, REINECKER H C, et al. A dense network of dendritic cells populates the murine epididymis[J]. Reproduction, 2011, 141(5): 653-663. DOI: 10.1530/REP-10-0493.

[24] BARRACHINA F, OTTINO K, TU L J, et al. CX3CR1 deficiency leads to impairment of immune surveillance in the epididymis[J]. Cell Mol Life Sci, 2022, 80(1): 15. DOI: 10.1007/s00018-022-04664-w.

[25] HERBERT J A, DENG Y, HARDELID P, et al. β₂-integrin LFA1 mediates airway damage following neutrophil transepithelial migration during respiratory syncytial virus infection[J]. Eur Respir J, 2020, 56(2): 1902216. DOI: 10.1183/13993003.02216-2019.

[26] CAREY R M, SIRAGY H M, GILDEA J J, et al. Angiotensin type-2 receptors: transducers of natriuresis in the renal proximal tubule[J]. Int J Mol Sci, 2022, 23(4). DOI: 10.3390/ijms23042317.

基金资助

国家自然科学基金(31971070)

陕西省重点研发计划一般项目(2022SF-241)