人类趋化素样因子超家族2参与小鼠精子形成

doi:10.19723/j.issn.1671-167X.2019.02.005

摘要/Abstract

摘要：

目的: 探究人类趋化素样因子超家族2(CKLF-like MARVEL transmembrane domain-containing protein 2, CMTM2)在小鼠精子生成过程中的作用。方法: 构建CMTM2基因敲除小鼠模型,利用Northern、RT-PCR及 Western 印迹检测野生型、杂合子及纯合子小鼠的CMTM2表达水平,统计小鼠生育幼崽数量及生育率,采集并分析精子活动度、形态及睾丸组织切片,检验血清睾酮及促卵泡激素(follicle-stimulating hormone, FSH)水平。结果: CMTM2在小鼠生精中呈现准确的调控式高度表达,基因敲除的成年小鼠和野生型成年小鼠在体重上差异没有统计学意义。纯合子的小鼠睾丸体积及重量小于野生型小鼠睾丸,野生型小鼠与CMTM2敲除小鼠的睾丸长径比较,差异有统计学意义[(11.32±1.21) mm vs. (8.29±1.92) mm, P<0.05],睾丸重量比较,差异有统计学意义[(101.63±2.33) mg vs. (85.22±2.84) mg, P <0.05]。与野生型小鼠相比,杂合子精子数量明显减少,纯合子小鼠精子发生停滞。在精子形态学方面,与野生型小鼠相比,杂合子小鼠有更多圆形精子,而纯合子则因精子发育停滞,多为圆形精子细胞。睾酮和FSH在3组间差异无统计学意义。纯合子雄性小鼠由于精子发生停滞而不育,纯合子小鼠缺失生精上皮层。结论: CMTM2在生精过程中发挥着不可或缺的作用,其参与精子发生的潜在机制有待于进一步实验研究以证实。

关键词: 人类趋化素样因子超家族2, 精子, 基因敲除小鼠模型, 精子生成

Abstract:

Objective: To investigate whether CKLF-like MARVEL transmembrane domain-containing protein 2 (CMTM2) is involved in spermatogenesis in mice. CMTM2 is highly expressed in testis, and could possibly be a potential spermagogenesis specific gene.Methods: CMTM2-deficient mouse model was generated. Northern, RT-PCR and Western blotting analysis were performed on total RNA derived from wild-type (WT, CMTM2 ^+/+) and CMTM2 ^+/- (heterozygote) and CMTM2 ^-/-(homozygote) mice to examine the CMTM2 level. The number of litters and the number of pups were counted and pregnancy rates calculated. The motility and morphology of the sperm and the histology of testes were analyzed. Se-rum testosterone and FSH concentrations were also measured. Standard t-tests were used and standard error of means were calculated.Results: CMTM2 was highly expressed in a finely regulated pattern in the mouse testis during spermatogenesis. The body weight of adult mice with CMTM2 deficiency was not significantly different from that of wild type mice. No obvious anatomical or behavioral abnormalities were observed. The testis of CMTM2 ^-/- was smaller than that of CMTM2 ^+/+ mice. The testis diameter in wild mice and CMTM2 null mice were (11.32±1.21) mm vs. (8.29±1.92) mm (P<0.05), and the weights were (101.63±2.33) mg vs. (85.22±2.84) mg (P<0.05), respectively. Female CMTM2 null mice were fertile, indicating that CMTM2 was not required for female gametogenesis. The CMTM2 ^-/- mice produced virtually no sperm, and CMTM2 ^+/- mice sperm count showed a significant decline. In terms of sperm morphorlogy study, more round spermatids could be observed in the heterozygote group, compared with the wild type group; while in the homozygote group, a large amount of round spermatids could be observed because of complete arrest of spermiogenesis. The hormone levels were not significantly different. The CMTM2 ^-/- male mice were sterile due to a late, complete arrest of spermiogenesis. The organized architecture of the seminiferous epithelium of the seminiferous tubules seen in CMTM2 ^+/+ mice was lost in CMTM2 ^-/- mice. Conclusion: This study suggests CMTM2 is not required for embryonic development in the mouse but is essential for spermiogenesis, however, further studies are required for more detailed mechanism study.

Key words: CMTM2, Sperm, Gene knockout mouse model, Spermiogenesis

中图分类号:

R698

张晓威,殷华奇,李清,赵永平,KiteBrandes,白文俊,徐涛. 人类趋化素样因子超家族2参与小鼠精子形成[J]. 北京大学学报（医学版）, 2019, 51(2): 228-233.

Xiao-wei ZHANG,Hua-qi YIN,Qing LI,Yong-ping ZHAO,BRANDES Kite,Wen-jun BAI,Tao XU. CMTM2 is involved in spermiogenesis in mice[J]. Journal of Peking University(Health Sciences), 2019, 51(2): 228-233.

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Genotype	Age/weeks	Testes weight/mg	Sperm count /(×10⁷/mL)	Motility/%	Morphology
CMTM2^+/+	8-12	101.63±2.33	3.28±0.34	86.33±2.98	Normal
CMTM2^+/-	8-12	98.87±2.76	2.07±0.57	33.45±3.67^*	Many round sperms
CMTM2^-/-	8-12	85.22±2.84^*	0^*	0^*	0^*