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Journal of Peking University(Health Sciences) ›› 2019, Vol. 51 ›› Issue (2): 228-233. doi: 10.19723/j.issn.1671-167X.2019.02.005

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CMTM2 is involved in spermiogenesis in mice

Xiao-wei ZHANG1,Hua-qi YIN1,Qing LI1,Yong-ping ZHAO2,BRANDES Kite3,Wen-jun BAI1,Tao XU1,∆()   

  1. 1. Department of Urology, Peking University People’s Hospital, Beijing 100044, China;
    2. Reproductive Medicine Center, Peking University People’s Hospital, Beijing 100044, China;
  • Received:2017-03-11 Online:2019-04-18 Published:2019-04-26
  • Contact: Tao XU E-mail:xutao@pkuph.edu.cn
  • Supported by:
    National Natural Foundation of China(81472393);Beijing Municipal Natural Science Foundation(7194327)

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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

CLC Number: 

  • R698

Figure 1

Genotype of some mice by PCR method M, marker (100 bp DNA ladder); N, negative control; KO, homozygote; HET, heterozygote; WT, wild type."

Figure 2

Analysis of testis gene expression in CMTM2 mutant mice A, northern blot analysis, the RNA from the mice whose genotype is shown above each lane was hybridized with a 32 P-labeled probe to detect the expression of the gene shown below each panel. The CMTM2 gene expression is abolished in CMTM2-/- animals; B, Western blot analysis, testes of CMTM2+/+, CMTM2+/- and CMTM2-/- were tested for CMTM2 protein expression. There was a dramatic reduction in the expression of CMTM2 in mutant mice. GAPDH was used as the loading control."

Figure 3

Comparison of the testis volume in CMTM2 mutant mice The testes of CMTM2-/- mice are smaller than those of CMTM2+/+."

Table 1

The comparison of testis weight, sperm count and motion parameters in different groups"

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*

Table 2

The mating experiment in different groups"

Male mice genotype Female mice Plugged mice Pregnant mice Offspring (M ∶F) AOA FCP/% FC/%
CMTM2+/+ (n=5)
CMTM2+/- (n=5)
CMTM2-/-(n=5)		 WT (n=49)
WT (n=51)
WT (n=50)		 38
34
36		 31
13
0*		 249 (132 ∶117)
103 (50 ∶53)
0*		 8.0
7.9
0*		 77.6
66.7
72.0		 63.3
25.5
0*

Figure 4

Testis histology in CMTM2 mutant mice A, histological staining of testis with hematoxylin and eosin (HE), and nearly complete germ cell loss is apparent in CMTM2-/- testis, whereas CMTM2+/+, CMTM2+/- testes look normal; B, spermatogonial depletion CMTM2-/- mice, CMTM2+/- mice look normal in comparison with CMTM2+/+ animals, whereas a progressive germ cell loss is also could be observed."

Table 3

The comparison of spermatids in seminiferous tubule in different groups"

Genotype SG PSC SSC Spermatids Sperms Sertoli cells
CMTM2+/+(n=5)
CMTM2+/- (n=5)
CMTM2-/- (n=5)		 39.11±2.98
37.73±4.55
14.22±5.01*		 67.92±1.03
63.34±6.01
20.18±8.82*		 2.96±0.73
2.29±0.74
0.00±0.00*		 139.22±7.81
128.18±8.17
30.84±13.89*		 40.84±5.92
29.28±3.44
0.00±0.00*		 9.21±1.29
9.45±1.78
9.01±1.54
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