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. 2020 Nov 3;10(1):18883.
doi: 10.1038/s41598-020-75957-y.

Dlec1 is required for spermatogenesis and male fertility in mice

Yu Okitsu  1 , Mamoru Nagano  1 , Takahiro Yamagata  2 , Chizuru Ito  3 , Kiyotaka Toshimori  3 , Hideo Dohra  4 , Wataru Fujii  5 , Keiichiro Yogo  6   7   8
Affiliations

Dlec1 is required for spermatogenesis and male fertility in mice

Yu Okitsu et al. Sci Rep. .

Abstract

Deleted in lung and esophageal cancer 1 (DLEC1) is a tumour suppressor gene that is downregulated in various cancers in humans; however, the physiological and molecular functions of DLEC1 are still unclear. This study investigated the critical role of Dlec1 in spermatogenesis and male fertility in mice. Dlec1 was significantly expressed in testes, with dominant expression in germ cells. We disrupted Dlec1 in mice and analysed its function in spermatogenesis and male fertility. Dlec1 deletion caused male infertility due to impaired spermatogenesis. Spermatogenesis progressed normally to step 8 spermatids in Dlec1-/- mice, but in elongating spermatids, we observed head deformation, a shortened tail, and abnormal manchette organization. These phenotypes were similar to those of various intraflagellar transport (IFT)-associated gene-deficient sperm. In addition, DLEC1 interacted with tailless complex polypeptide 1 ring complex (TRiC) and Bardet-Biedl Syndrome (BBS) protein complex subunits, as well as α- and β-tubulin. DLEC1 expression also enhanced primary cilia formation and cilia length in A549 lung adenocarcinoma cells. These findings suggest that DLEC1 is a possible regulator of IFT and plays an essential role in sperm head and tail formation in mice.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Expression analysis of mouse DLEC1. (A–C) Analysis of Dlec1, Vill, and Ctdspl expression using RT-PCR. Expression in (A) various mouse tissues, (B) WT and W/WV mutant mouse testes, and (C) testes during first-wave spermatogenesis. The numbers indicate days after birth. (D) DLEC1 expression during postnatal testicular development was examined by western blotting. The numbers indicate days after birth. (E) DLEC1 expression in testes, cauda epididymis, and cauda epididymal sperm. 1% Triton X-100-containing lysis buffer (T) or 0.1% SDS-containing RIPA buffer (R) was used to solubilize the tissue and use for western blotting. Images of full-length gels and immunoblots are presented in the supplementary Fig. S6.
Figure 2
Figure 2
Dlec1 is required for spermatogenesis and male fertility. Each male mouse was caged with two WT BDF1 female mice for 2 weeks, and the (A) pregnancy rate (number of pregnant females/number of females mated) and (B) average of litter size were measured (n = 6). (C) Appearance of testes and epididymides of WT and Dlec1−/− mice. (D) Testis weight per body weight of WT and Dlec1−/− mice (n = 6). No significant statistical difference between WT and Dlec1−/− mice was detected (p = 0.066). (E) Sections of testis (a–d) and cauda epididymis (e and f) were stained using H&E. *Seminiferous tubule where sperm flagella were observed. (c and d) Magnified areas of the boxes in (a) and (b), respectively. Scale bar = 100 µm. (F) Number of cauda epididymal sperm in WT and Dlec1−/− mice (n = 5). (G) Morphology of WT and Dlec1−/− sperm. Scale bar = 10 µm. (H) Percentage of motile sperm of WT and Dlec1−/− sperm (n = 5). Error bars indicate standard error. (I) The testis section was stained using anti-acetylated tubulin antibody (green) and DAPI (blue). No acetylated tubulin signal was observed in the seminiferous tubule lumen in Dlec1−/− testes. Scale bar = 100 µm.
Figure 3
Figure 3
Spermatogenesis is impaired at the elongating spermatid stage in Dlec1−/− mice. (A) Seminiferous epithelium in WT and Dlec1−/− mouse testis. Testis sections were stained using periodic acid solution and haematoxylin. Roman numerals indicate stages of the cycle. (B) Spermatids at each stage of spermatogenesis in WT and Dlec1−/− mice. Testis sections were stained as in (A). Numerals indicate the stages of spermatids. (C) Acrosome formation in WT and Dlec1−/− spermatids. Isolated spermatids (step 7–12) were stained with Alexa488-conjugated peanut agglutinin (PNA, green). (D–K) Electron microscopy of WT and Dlec1−/− testis cross sections. (D) Seminiferous tubule at stage VI in WT testis. Arrows and arrowheads indicate round spermatids and flagella cross sections, respectively. (E) Seminiferous tubule at stage VII in Dlec1−/− testis. Although round spermatids are normal (arrow), many degenerated cells and irregular cellular components were observed (arrowhead). (F–I) Elongating spermatids (step 11 and 12) in (F,H) WT and (G,I) Dlec1−/− testes at (F,G) stage XI and (H,I) stage XII. The head shape was deformed in Dlec1−/− spermatids. (J) Abnormal tail-like structure in Dlec1−/− spermatids. Misaligned mitochondria (arrowhead) and fibre-like structures (arrow) were observed. (K) Phagocytosis of degenerated spermatids by Sertoli cells. Digested cellular components in the phagosome (arrowhead) and incorporation of the fibre-like structure (arrow) were observed.
Figure 4
Figure 4
The manchette is abnormally elongated in Dlec1−/− spermatids. Testicular cells were isolated and stained using anti-α-tubulin antibody (mouse monoclonal, green). Photographs of each step of spermatids of WT and Dlec1−/− mice were taken. Rs, round spermatids. Nuclei were stained using DAPI (blue).
Figure 5
Figure 5
DLEC1 interacts with tubulin, TRiC subunits, and BBSome subunits. 3 × FLAG-tagged human DLEC1 (FLAG-hDLEC1) or empty vector was expressed in HEK293F cells and immunoprecipitated using anti-FLAG antibody. SDS-PAGE was applied to the immunoprecipitates, followed by western blotting using (A) tubulin antibodies, (B) TRiC subunit antibodies, and (C) BBSome subunit antibodies. WCL, whole cell lysate. Images of full-length immunoblots are presented in the supplementary Fig. S7.
Figure 6
Figure 6
DLEC1 expression enhances primary cilia formation. (A) Upper panel: Tubulin polymerization assay. FLAG-hDLEC1 was introduced into HEK293F cells, and the cells were lysed with hypotonic buffer. After centrifugation, supernatants (S) and precipitates (P) were collected and used for western blotting. Nocodazole (Noc) and paclitaxel (Pac), which induce microtubule destabilization and stabilization, respectively, were used as a control experiment. Lower panel: DLEC1 expression was confirmed by western blotting. (B) Quantification results of tubulin polymerization assay shown in (A). P/S indicates ratio of signal intensity of precipitates (P) and supernatants (S). Data are presented as average ± standard error (n = 3). (C) Tubulin polymerization in WT and Dlec1−/− testicular cells was monitored as in (A). (D) Establishment A549 cells stably express human DLEC1. Empty vector or 3 × FLAG-tagged human DLEC1 expression vector were introduced into A549 cells and selected by puromycin. Expression of DLEC1 was monitored by western blotting using anti-human DLEC1 antibody. (E) Empty vector or hDLEC1 expressing A549 cells grown to confluent were serum-starved for 24 h and stained with anti-ARL13B antibody, a primary cilia marker. Scale bar = 10 μm. (F) Percentage of cells with cilia were quantified. Over 500 cells in one experiment were counted, and data were shown as average ± standard error (n = 3). Asterisk indicates a significant difference (Student’s t test, p < 0.05). (G) Ciliogenesis was induced as described in (E) and cilia length were measured. Data were counted from approximately 300 cilia from 3 independent experiments and shown in boxplots. Double asterisk indicates significant differences (Student’s t test, p < 0.01). (H) Intracellular localisation of hDLEC1 in A549 cells. 3 × FLAG-tagged hDLEC1 expressing A549 cells were immunostained with anti-ARL13B and anti-FLAG antibodies. Images of full-length immunoblots are presented in the supplementary Fig. S8.
Figure 7
Figure 7
Expression, complex formation, and intracellular localisation of TRiC and BBSome is normal in Dlec−/− testis. (A) TRiC and BBSome subunit expression in WT and Dlec1−/− testes was monitored by western blotting. (B) Complex formation of TRiC and BBSome in WT and Dlec1−/− testes. Testis lysates were layered onto a 10–40% sucrose gradient and centrifuged at 28,000 rpm for 16 h. The fractions were recovered and western blotting applied. Positions of molecular weight standards are indicated at the top. (C) Intracellular localisation of TCP-1 α, BBS2, IFT25, and IFT140 in WT and Dlec1−/− round (upper) and elongating spermatids (lower). Scale bar = 10 μm. Images of full-length immunoblots are presented in the supplementary Fig. S9.
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References

    1. Mäkelä JA, Toppari J. Spermatogenesis. In: Simoni M, Huhtaniemi I, editors. Endocrinology of the Testis and Male Reproduction. New York: Springer; 2017. pp. 1–39.
    1. Shima JE, McLean DJ, McCarrey JR, Griswold MD. The murine testicular transcriptome: characterizing gene expression in the testis during the progression of spermatogenesis. Biol. Reprod. 2004;71:319–330. doi: 10.1095/biolreprod.103.026880. - DOI - PubMed
    1. Schultz N, Hamra FK, Garbers DL. A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets. Proc. Natl. Acad. Sci. U.S.A. 2003;100:12201–12206. doi: 10.1073/pnas.1635054100. - DOI - PMC - PubMed
    1. Pleuger C, Lehti MS, Dunleavy JE, Fietz D, O'Bryan MK. Haploid male germ cells—the Grand Central Station of protein transport. Hum. Reprod. Update. 2020 doi: 10.1093/humupd/dmaa004. - DOI - PubMed
    1. Porter ME, Sale WS. The 9 + 2 axoneme anchors multiple inner arm dyneins and a network of kinases and phosphatases that control motility. J. Cell Biol. 2000;151:F37–42. doi: 10.1083/jcb.151.5.f37. - DOI - PMC - PubMed

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