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. 2023 Apr 26;12(9):1264.
doi: 10.3390/cells12091264.

Zbtb40 Deficiency Leads to Morphological and Phenotypic Abnormalities of Spermatocytes and Spermatozoa and Causes Male Infertility

Yinghong Cui  1   2   3 , Mingqing Zhou  1 , Quanyuan He  1 , Zuping He  1   2   3
Affiliations

Zbtb40 Deficiency Leads to Morphological and Phenotypic Abnormalities of Spermatocytes and Spermatozoa and Causes Male Infertility

Yinghong Cui et al. Cells. .

Abstract

Studies on the gene regulation of spermatogenesis are of unusual significance for maintaining male reproduction and treating male infertility. Here, we have demonstrated, for the first time, that a loss of ZBTB40 function leads to abnormalities in the morphological and phenotypic characteristics of mouse spermatocytes and spermatids as well as male infertility. We revealed that Zbtb40 was expressed in spermatocytes of mouse testes, and it was co-localized with γH2AX in mouse secondary spermatocytes. Interestingly, spermatocytes of Zbtb40 knockout mice had longer telomeres, compromised double-strand break (DSB) repair in the sex chromosome, and a higher apoptosis ratio compared to wild-type (WT) mice. The testis weight, testicular volume, and cauda epididymis body weight of the Zbtb40+/- male mice were significantly lower than in WT mice. Mating tests indicated that Zbtb40+/- male mice were able to mate normally, but they failed to produce any pups. Notably, sperm of Zbtb40+/- mice showed flagellum deformities and abnormal acrosome biogenesis. Furthermore, a ZBTB40 mutation was associated with non-obstructive azoospermia. Our results implicate that ZBTB40 deficiency leads to morphological and phenotypic abnormalities of spermatocytes and spermatids and causes male infertility. This study thus offers a new genetic mechanism regulating mammalian spermatogenesis and provides a novel target for gene therapy in male infertility.

Keywords: ZBTB40 mutation; Zbtb40 knockout; male infertility; spermatids; spermatocytes; telomere length.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
The expression of ZBTB40 in mouse testes. (A) Immunohistochemistry showed the expression of Zbtb40 in mouse spermatocytes. (B) Double immunofluorescence revealed the co-localization of Zbtb40 with γH2AX in mouse spermatocytes. Right panel is the enlargement of areas in left panel. White arrows indicated the spermatocytes that were positive for Zbtb40 and γH2AX.
Figure 2
Figure 2
The phenotypical characteristics of Zbtb40+/− male mice. (A) Testes (left panel) and cauda epididymis (right panel) of the Zbtb40+/−#4 mice and the WT mice. (B–D) Comparison of testicular volume (B), cauda epididymis and body weight ratio (C), and testis/body weight ratio (D) of the 5-month-old Zbtb40+/− male mice and the WT mice. (E) Sperm counts were reduced in the Zbtb40+/− mice compared to the WT mice. (F) Motile sperm was decreased in the Zbtb40+/− mice compared to the WT mice. For p values in (B–F) * indicated p < 0.05, *** implicated p < 0.001, n = 3. (G) H&E staining demonstrated abnormal morphology of testes and cauda epididymis of the Zbtb40+/− mice. (H) Representative pictures of testes in the Zbtb40+/− mice and WT mice. Red arrows indicated pyknotic spermatocytes.
Figure 3
Figure 3
The influence of Zbtb40+/− on apoptosis and the proliferation of mouse male germ cells. (A,B) TUNEL measured percentages of apoptosis between Zbtb40+/− mice and the WT mice. (C,D) Immunohistochemistry showed Ki67-positive germ cells between Zbtb40+/− mice and the WT mice. For p values of B and D: ** denoted p < 0.01, n = 3.
Figure 4
Figure 4
The defective phenotypes of spermatocytes of Zbtb40+/− mice. (A) Representative spermatocyte spreading revealed the abnormal XY dissociation in Zbtb40+/−(−400bp) mice and the WT mice. The white arrows denote sex chromosome. (B) The bar chart presents the rates of XY separation in samples of Zbtb40+/− mice and the WT mice. For p values in B * indicated p < 0.05.
Figure 5
Figure 5
The sperm of Zbtb40+/− male mice show flagellum deformities. (A) Pap stain was used to observe sperm morphology of the Zbtb40+/− mice and the WT mice. (B) Representative photos of abnormal sperm of the Zbtb40+/− mice. (C) The bar chart indicates the rates of abnormal sperm in samples of the Zbtb40+/− mice. (D) TEM of the cross-sections of sperm flagella displayed abnormal axoneme and peri-axoneme structures of the Zbtb40+/− mice. ODFs: outer dense fibers; DMT: outer doublet microtubules; MS: mitochondrial sheath; CP: central pair complex. (E) Immunofluorescence was used to observe expression of α-tubulin in sperm tails of the Zbtb40+/− mice. (F) The bar chart shows the rates of abnormal α-tubulin in the Zbtb40+/− mice. For p values of (C,F): * indicated p < 0.05, n = 3.
Figure 6
Figure 6
The sperm of Zbtb40+/− male mice assume abnormal acrosome biogenesis. (A) TEM was used to detect the acrosome biogenesis in the Zbtb40+/− mouse. Red arrows indicate abnormal acrosome. (B) Immunofluorescence was used to detect the expression of FITC-PSA in sperm head of the Zbtb40+/− mice. The white arrows denote flawed acrosome. (C) The bar chart displays the rates of acrosomal integrity in the Zbtb40+/− mice. For p values of t-tests in (C): * indicated p < 0.05, n = 3.
Figure 7
Figure 7
Zbtb40+/− mice have longer telomere length. (A) Representative spermatocytes were separated by STA-PUT from the Zbtb40+/− mice. (B) Q-FISH showed telomere foci of spermatocytes in the Zbtb40+/− mice and the WT mice. (C) The distribution of relative telomere length in the Zbtb40+/− mice and the WT mice. For p values of t-tests in (C): **** denoted p < 0.0001.
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