Roles of Cep215/Cdk5rap2 in establishing testicular architecture for mouse male germ cell development

Abstract

Cep215/Cdk5rap2 is a centrosome protein crucial for directing microtubule organization during cell division and morphology. Cep215 is a causal gene of autosomal recessive primary microcephaly type 3, characterized by a small brain size and a thin cerebral cortex. Despite previous attempts with Cep215 knockout (KO) mice to elucidate its developmental roles, interpreting their phenotypes remained challenging due to potential interference from alternative variants. Here, we generated KO mice completely lacking the Cep215 gene and investigated its specific contributions to male germ cell development. In the absence of Cep215, testis size decreased significantly, accompanied by a reduction in male germ cell numbers. Histological analyses unveiled the arrested development of male germ cells around the zygotene stage of meiosis. Concurrently, the formation of the blood-testis barrier (BTB) was impaired in Cep215 KO testes. These findings suggest that BTB failure contributes, at least partially, to male germ cell defects observed in Cep215 KO mice. We propose that the deletion of Cep215 may disrupt microtubule organization in Sertoli cells with a delay in spermatogonial stem cell mitosis, thereby impeding proper BTB formation.

Keywords: Cep215/Cdk5rap2; Sertoli cells; blood‐testis barrier; male germ cells; meiosis; testis.

FIGURE 1

Generation of the Cep215 total KO mouse. (A) Summary of a total deletion of the Cep215 gene using the CRISPR‐Cas9 system. The guide RNAs targeted introns 1 and 34, resulting in a deletion of 175 kb from exons 2–34 of the Cep215 gene. (B) PCR genotyping of the Cep215 KO mice. (C) Immunoblot analyses of the tissues from adult Cep215 KO mice with Cep215 and Gapdh antibodies. (D) The number of progenies from Cep215 heterozygote matings. (E) Cep215 KO and littermate (WT) mice at P17. (F) The body weights of the adult Cep215 KO and littermate (WT) mice. (G–I) H&E staining of the whole brain of Cep215 KO mice at P17. The cortical thickness (black arrows) and the length (white arrows) of the brain (G) were marked. Scale bars: 1 mm. The cortical thickness (H) and the length (I) of the brains were measured. (F, H, I) Values are means and SEM (n = 3). The statistical significance was determined by unpaired t‐test. *p < .05.

FIGURE 2

Histological analysis of the Cep215 KO testes. (A) Testes of the Cep215 KO and littermate (WT) mice at P17. (B) Weights of the testes from the Cep215 KO mice at P17. (C) H&E staining of the testes from Cep215 KO mice at P7, P17, and adult. Scale bars: 50 μm. (D) The number of seminiferous tubules with spermatogonia (Sg), spermatocytes (Sc), and spermatids (Tid) was counted. At least 200 tubules per experimental group were analyzed (n = 3). (B, D) Values are means and SEM. The statistical significance was determined by unpaired t‐test (B) or two‐way ANOVA (D). *p < .05.

FIGURE 3

The numbers of germ, Sertoli, and Leydig cells in Cep215 KO testes. (A) Immunohistochemistry of the testes from Cep215 KO mice at P7 and P17 with Sox9 (green) and γH2AX (red) antibodies. (B) The number of Sertoli and germ cells per tubule was counted. At least 1200 cells per experimental group were counted (n = 3). (C) Immunohistochemistry of the testes from Cep215 KO mice at P7, P17, and adult with HSD3β antibody. (D) The number of Leydig cells per section was counted (bars). The cross‐sectional area was also measured (dots). At least 90 cells per section were counted (n = 3). (A, C) Scale bars: 50 μm. (B, D) Values are means and SEM. The statistical significance was determined by two‐way ANOVA (B) and unpaired t‐test (D). *p < .05. ns, not significant.

FIGURE 4

Phenotypes of the Cep215;Trp53, double KO mice. (A) Genomic PCR analyses for genotyping of the Cep215;Trp53 double KO mice. (B) Brains and testes of the Cep215;Trp53 double KO mice. Scale bars: 5 mm. (C) H&E staining of the brains from Cep215;Trp53 double KO mice. The cortical thickness (black lines) and the length (white arrows) of the brain were marked. Scale bar, 1 mm. (D, E) The cortical thickness (D) and length (E) of the brains were measured. (F) H&E staining of the testes from adult Cep215;Trp53 double KO mice. Scale bar, 50 μm. (G) The number of seminiferous tubules with spermatogonia (Sg), spermatocytes (Sc), and spermatids (Tid) was counted. At least 170 tubules per experimental group were analyzed in two mice. (D, E, G) Values are means and SEM. ns, not significant.

FIGURE 5

Meiotic defects in the Cep215 KO male germ cells. (A) Immunohistochemistry of the testes from Cep215 KO mice at P7, P17, and adult with γH2AX antibody (green). (B) The number of tubules with γH2AX‐positive meiotic cells was counted. At least 220 tubules per experimental group were counted. (C) TUNEL assays with the testes of Cep215 KO mice at P7, P17, and adult. (D) The number of TUNEL‐positive spermatogonia (Sg) and spermatocytes (Sc) was counted. At least 1000 cells per experimental group were counted. (E) Co‐immunostaining of the spermatocytes isolated from the testes of adult Cep215 KO mice with the Sycp3 (green) and γH2AX (red) antibodies. Meiotic stages were determined based on the immunostaining patterns. Scale bars: 10 μm. (F) The number of spermatocytes at specific meiotic stages was counted. At least 270 cells per experimental group were counted (n = 3). (A, C, E) Nuclei were stained with DAPI (blue). (A, C) Scale bars, 50 μm. (B, D, F) Values are means and SEM. The statistical significance was determined by two‐way ANOVA. *p < .05, compared to the wild type groups.

FIGURE 6

Centrosomes in the Cep215 KO male germ cells. (A) Immunohistochemistry of the Cep215 KO testes with Cep215 (green), centrin‐2 (red), Pcnt (green), and γ‐tubulin (red) antibodies. Inlets are enlarged views of the male germ cells at specific developmental stages (Sg, spermatogonia; Sc spermatocyte; RT, round spermatid; ET, elongated spermatid). Scale bars, 50 μm. (B) Immunostaining of the isolated spermatogonia and spermatocytes from Cep215 KO testes with Cep215 (red), centrin‐2 (green), Pcnt (red) and γ‐tubulin (green) antibodies. Scale bars, 10 μm. (A, B) Nuclei were stained with DAPI (blue). (C–F) Centrosome intensities of Cep215 (C), centrin‐2 (D), Pcnt (E), and γ‐tubulin (F) in the Cep215 KO spermatocytes at P17. At least 50 cells per experimental group were counted (n = 3). Fluorescent intensities were displayed with box‐and‐whiskers plots (lines, median; vertical boxes, values from 25th and 75th; down error bars, 5th value, up error bar, 95th value; circle, outliers). The statistical significance was determined by an unpaired t‐test. *p < 0.05. ns, not significant.

FIGURE 7

Centrosomes in the Cep215 KO Sertoli cells. (A) Immunohistochemistry of the Cep215 KO testes at P17 with Cep215 (red), centrin‐2 (green), Pcnt (red), γ‐tubulin (green), and vimentin (cyan) antibodies. Inlets are enlarged views of the Sertoli cells marked with the vimentin antibody. (B) Immunostaining of the isolated Sertoli cells with the Cep215 (red), centrin‐2 (green), Pcnt (red), and γ‐tubulin (green) antibodies. (A, B) Nuclei were stained with DAPI (blue). Scale bars: 20 μm. (C–F) Centrosomal intensities of Cep215 (C), centrin‐2 (D), Pcnt (E), and γ‐tubulin (F) in Sertoli cells. At least 100 cells per experimental group were counted (n = 3). Fluorescent intensities were displayed with box‐and‐whiskers plots (lines, median; vertical boxes, values from 25th and 75th; down error bars, 5th value, up error bar, 95th value; circle, outliers). The statistical significance was determined by an unpaired t‐test. *p < .05. ns, not significant.

FIGURE 8

Failure of the blood–testis barrier (BTB) in the Cep215 KO testes. (A) The biotin‐penetration assays with the Cep215 KO mice at indicated ages. (B) The number of biotin‐penetrated seminiferous tubules was counted. More than 100 tubules per experimental group were analyzed. (C) Immunohistochemistry of the testes of Cep215 KO mice at P17 with connexin‐43 (green), claudin‐11 (green), and γH2AX (red) antibodies. Nuclei were stained with DAPI (blue). (D) The number of tubules containing pachytene spermatocytes was counted. (E, F) The number of tubules with intact BTB structure was counted. The intactness of BTB was determined with the immunostaining staining patterns of connexin‐43 (E) and claudin‐11 (F). At least 200 tubules per experimental group were analyzed (n = 3). (A, C) Stars represent seminiferous tubules with intact BTB. Scale bars: 50 μm. (B, D, E, F) Values are means and SEM. The statistical significance was determined by two‐way ANOVA. *p < .05, compared to the wild type.

FIGURE 9

Model in immature testes, seminiferous tubules comprise spermatogonial stem cells and Sertoli cells. During puberty in mice, Sertoli cells undergo polarization and establish interactions with neighboring Sertoli cells, culminating in the formation of BTB. This process involves the rearrangement of microtubules parallel to the polarity of Sertoli cell, facilitating proper cell–cell interactions and barrier formation. In Cep215 KO testes, the proliferation activity of the spermatogonial stem cells decreases and Sertoli cells fail to polarize, possibly due to microtubule misorientation. Consequently, BTB is not formed and spermatogenesis is blocked prior to meiosis in Cep215 KO mice.