Cdc42 activity in Sertoli cells is essential for maintenance of spermatogenesis

Abstract

Sertoli cells are highly polarized testicular supporting cells that simultaneously nurture multiple stages of germ cells during spermatogenesis. Proper localization of polarity protein complexes within Sertoli cells, including those responsible for blood-testis barrier formation, is vital for spermatogenesis. However, the mechanisms and developmental timing that underlie Sertoli cell polarity are poorly understood. We investigate this aspect of testicular function by conditionally deleting Cdc42, encoding a Rho GTPase involved in regulating cell polarity, specifically in Sertoli cells. Sertoli Cdc42 deletion leads to increased apoptosis and disrupted polarity of juvenile and adult testes but does not affect fetal and postnatal testicular development. The onset of the first wave of spermatogenesis occurs normally, but it fails to progress past round spermatid stages, and by young adulthood, conditional knockout males exhibit a complete loss of spermatogenic cells. These findings demonstrate that Cdc42 is essential for Sertoli cell polarity and for maintaining steady-state sperm production.

Keywords: Cdc42; Rho GTPase; Sertoli cell; blood testis barrier; cell junction; cell polarity; ectoplasmic specialization; spermatogenesis.

Figure 1.. Sertoli-cell-specific ablation of Cdc42 causes complete loss of germ cells in the adult testis

(A) Image showing adult (P60) testis size decrease in cKO (Dhh-Cre;Cdc42^flox/flox) testes versus Dhh-Cre;Cdc42^flox/+ heterozygous control testes. (B and C) Graphs showing average testis weight (B) and average testis weight:body weight ratio (C) of P60 control Dhh-Cre;Cdc42^flox/+ males versus cKO males (n = 6 control and n = 4 cKO testes, each from independent males). (D and E) Graphs showing average tubule cross-sectional area (D) and average number of SOX9⁺ cells per tubule (E) of P90 control (Dhh-Cre;Cdc42^flox/+) versus cKO tubules (n = 15 tubules from each of 3 independent males). (F–I) Immunofluorescence images of P60 (F and G) and P90 (H and I) adult control Dhh-Cre;Cdc42^flox/+ (F and H) and cKO (G and I) testes, showing absence of TRA98⁺ germ cells in cKO testes. (F’)–(I’) are higher-magnification images of the boxed regions in (F)–(I). Dashed lines indicate tubule boundaries. Scale bars, 100 μm. Data in (B)–(E) are shown as means ± SDs. p values were calculated via a 2-tailed Student’s t test.

Figure 2.. Cdc42 in Sertoli cells is dispensable for fetal testicular differentiation

Immunofluorescence images of E18.5 control Dhh-Cre;Cdc42^flow/+ (A, C, E, G, I, K, and M) and cKO (B, D, F, H, J, L, and N) testes. (A–D) Control (A and C) and cKO (B and D) testes contain similar numbers of germ (TRA98⁺) and Sertoli (AMH⁺/SOX9⁺) cells, and they display similar expression of CTNNB1 within tubules. (E-H) Both control (E and G) and cKO (F and H) Sertoli cells are in active cell cycle (MKI67⁺). (I and J) Both control (I) and cKO (J) germ cells are quiescent (MKI67⁻). (K–N) Control (K and M) and cKO (L and N) testes show similar numbers of vascular endothelial (PECAM1⁺), Leydig (CYP17A1⁺), and immune (CD45⁺) cells. Scale bars, 100 μm.

Figure 3.. Early postnatal differentiation of Sertoli and germ cells occurs normally in cKO testes.

Immunofluorescence images of P7 control Dhh-Cre;Cdc42^flox/+ (A, C, E, G, I, and K) and cKO (B, D, F, H, J, and L) testes. (A’)–(D’) are higher-magnification images of the boxed regions in (A)–(D). (A–H) Compared to P7 controls (A, C, E, and G), P7 cKO testes (B, D, F, and H) have similar numbers and localization of Sertoli cells (SOX9⁺, GATA4⁺), undifferentiated spermatogonia (GFRA1⁺, ZBTB16⁺), differentiating spermatogonia (KIT⁺), and overall germ cells (DDX4⁺, TRA98⁺). (I and J) GATA1 expression is detected in both control (I) and cKO (J) Sertoli cells. (K and L) Both AMH and AR are detected within control (K) and cKO (L) testes, but AMH expression appears slightly decreased in a subset of cKO Sertoli cells. Scale bars, 100 μm.

Figure 4.. Cdc42 is not required for onset and maintenance of Sertoli cell quiescence but is required for Sertoli cell survival

Immunofluorescent images of P60 (A–D), P90 (E and F), P24 (G, H, M, and N), P30 (I and J), and P15 (K and L) control Dhh-Cre;Cdc42^flox/+ (A, C, E, G, I, K, and M) and cKO (B, D, F, H, J, L, and N) testes. (A’)–(F’), (H’), (J’), and (K’)–(N”) are higher-magnification images of the boxed regions in (A)–(F), (H), (J), and (K)–(N). Dashed lines throughout the figure indicate tubule boundaries. (A and B) Both P60 control (A) and cKO (B) Sertoli cells are MKI67⁻, but rare cKO Sertoli cells expressing MKI67 (arrowheads in B’) are observed. (C and D) Neither control (C) or cKO (D) Sertoli cells express the mitotic marker pHH3 (arrowheads in C’ and D’); only germ cells in control tubules are pHH3⁺ (arrow in C’). (E and F) Cleaved caspase 3⁺ (apoptotic) Sertoli cells are rarely detected in P90 control (E) testes but are often observed in cKO (F) testes (arrowhead in F’). (G–J) Compared to control P24 (G) and P30 (I) juvenile testes, cKO juvenile testes (H and J) exhibit increased cleaved caspase 3⁺ Sertoli cells (GATA1⁺; arrow in H’) and germ cells (TRA98⁺; arrowheads in H’ and J’). (K–N) Relative to control P15 (K) and P24 (M) juvenile Sertoli cells, cKO juvenile Sertoli cells (L and N) show similar immunonegativity for MKI67 (arrowheads in L” and N”), apart from rare MKI67⁺ cells (arrow in N’). Scale bars, 100 μm. (O) Graph showing percentage of tubules containing cleaved caspase 3⁺ cells in P24 control versus cKO testes (n = 15 tubules each from 3 testes each for controls and cKO). Data are shown as means ± SDs. p value was calculated via a 2-tailed Student’s t test.

Figure 5.. Juvenile cKO mice show defects in Sertoli cell development

Immunofluorescent images of P24 (A, B, and F–K) control Dhh-Cre;Cdc42^flox/+ (A, F, H, and J) and cKO (B, G, I, and K) testes. (F’)–(I’) are higher-magnification images of the boxed regions in (F)–(I). (A and B) Relative to control testes (A), cKO testes (B) show smallertubules and mislocalization of Sertoli cell nuclei (red arrows in B) near the center of tubules. (C–E) Graphs showing average number of SOX9⁺ Sertoli cells per tubule (C), average number of TRA98⁺ germ cells per tubule (D), and average tubule cross-sectional area (E) in P24 control versus cKO testes (n = 15 tubules each from 3 testes each for controls and cKO). The data in (C)–(E) are shown as means ± SDs. p values were calculated via a 2-tailed Student’s t test. (F–I) Relative to control (F and H) testes that exhibit nuclear localization of AR and undetectable expression of AMH, cKO testes (G and I) show only weak or sporadic localization of AR in Sertoli cells (arrow in G’) and persistent expression of AMH. (J and K) Both control (J) and cKO (K) testes exhibit strong expression of GDNF. Scale bars, 100 μm.

Figure 6.. Loss of Cdc42 leads to disruptions in Sertoli cell polarity and BTB function in cKO juvenile testes

Immunofluorescent images of P24 control Dhh-Cre;Cdc42^flox/+ (A, C, E, G, I, and K), P30 control (M), P24 cKO (B, D, F, H, J, and L), and P30 cKO (N) testes. (M’) and (N’) are higher-magnification images of the boxed regions in (M) and (N). (A–H) While control (A, C, E, and G) testes showed compartment-specific enrichment of CLDN11, CTNNB1, PARD3, SCRIB, and phalloidin staining (arrowheads), cKO testes (B, D, F, and H) exhibited aberrant localization of these factors (arrows). (I–L) Both control (I and K) and cKO (J and L) testes displayed strong expression of ITGA6 and ITGB1 in the tubule basement membrane; however, cKO testes regularly displayed ectopic expression of ITGB1 in the central portion of tubules. (M and N) While P30 control (M) testes showed only the interstitial and basal tubular presence of biotin (detected by streptavidin staining) in a tracer assay, cKO testes (N) showed widespread presence of biotin deep within tubules (arrows in N’) (n = 3 testes each for controls and cKO). Dashed lines indicate tubule boundaries. Scale bars, 50 μm.

Figure 7.. Cdc42 in Sertoli cells is essential for progression past the round spermatid stage during first-wave spermatogenesis

Immunofluorescent images of P24 (A, B, E, F, I, J, L, and M) and P30 (N and O) control Dhh-Cre;Cdc42^flox/+ (A, E, I, L, and N) and cKO (B, F, J, M, and O) juvenile testes. (A’), (B’), (I’), (J’), and (L’)–(O’) are higher-magnification images of the boxed regions in (A), (B), (I), (J), and (L)–(O). (A and B) Both control (A) and cKO (B) testes contain basally localized GFRA1⁺ undifferentiated spermatogonia. (C) Graph showing average number of GFRA1⁺ cells within tubules in P24 control versus cKO testes (n = 23–104 tubules per testis from 3 testes each for controls and cKO). (D) Graph showing distribution of percentage of tubules containing 1–4 GFRA1⁺ cells in P24 control versus cKO testes (n = 13–50 tubules per testis from 3 testes each for controls and cKO). (E and F) Control (E) and cKO (F) testes both contain tubules with STRA8⁺ preleptotene spermatocytes (green asterisks), but cKO testes contain fewer ZBTB16⁺ cells. (G) Graph showing average number of ZBTB16⁺ cells per tubule in P24 control versus cKO testes (n = 15 tubules each from 3 testes each for controls and cKO). (H) Graph showing average percentage of tubules containing STRA8⁺ preleptotene spermatocytes in P24 control versus cKO testes (n = 58–156 tubules per testis from 3 testes each for controls and cKO). (I and J) Compared to control (I) testes, cKO testes (J) contain fewer KIT⁺ differentiating spermatogonia (also DDX4⁺; arrowheads); however, KIT expression in Leydig cells (arrows) is comparable between control and cKO testes. (K) Graph showing average number of KIT⁺ cells in P24 control versus cKO tubules (n = 15 tubules each from 3 testes for controls and 2 testes for cKO). (L and M) γ-H2AX⁺ leptotene (arrows) and zygotene spermatocytes, as well as H1F6⁺ (H1T⁺) pachytene spermatocytes with proper sex-body-enriched γ-H2AX localization (arrowheads), are observed in both control (L) and cKO (M) tubules; however, the number of H1F6⁺ spermatocytes is decreased in cKO tubules. (N and O) While P30 control (N) tubules contain round spermatids (green arrow in N’) and elongating spermatids (green arrowhead in N’), P30 cKO tubules only contain round spermatids (green arrow in O’). The red dashed line indicates tubule boundaries. Scale bars, 50 μm. All graph data are shown as means ± SDs. p values were calculated via a 2-tailed Student’s t test.