Distinct Roles for Rac1 in Sertoli Cell Function during Testicular Development and Spermatogenesis

Abstract

Sertoli cells are supporting cells of the testicular seminiferous tubules, which provide a nurturing environment for spermatogenesis. Adult Sertoli cells are polarized so that they can simultaneously support earlier-stage spermatogenic cells (e.g., spermatogonia) basally and later-stage cells (e.g., spermatids) apically. To test the consequences of disrupting cell polarity in Sertoli cells, we perform a Sertoli-specific conditional deletion of Rac1, which encodes a Rho GTPase required for apicobasal cell polarity. Rac1 conditional knockout adults exhibit spermatogenic arrest at the round spermatid stage, with severe disruption of Sertoli cell polarity, and show increased germline and Sertoli cell apoptosis. Thus, Sertoli Rac1 function is critical for the progression of spermatogenesis but, surprisingly, is dispensable for fetal testicular development, adult maintenance of undifferentiated spermatogonia, and meiotic entry. Our data indicate that Sertoli Rac1 function is required only for certain aspects of spermatogenesis and reveal that there are distinct requirements for cell polarity during cellular differentiation.

Keywords: RAC1; Sertoli cell; blood-testis barrier; cell polarity; ectoplasmic specialization; spermatogenesis.

Figure 1.. Sertoli Rac1 Function Is Required for Proper Adult Testicular Development

(A–J) Three-month-old (P90) control Dhh-Cre;Rac1^flox/+ (A, C, E, G, and I) and Dhh-Cre;Rac1^flox/flox (cKO) (B, D, F, H, and J) testes. (C’), (D’), (I’), and (J’) are higher-magnification images of the boxed regions in (C), (D), (I), and (J). (A and B) Relative to controls (A), Rac1 cKO testes (B) exhibit smaller tubules with varying numbers of TRA98⁺ germ cells. (C and D) Rac1 cKO testes (D) show GATA1 expression in all tubules, in contrast to heterogeneous expression in controls (C). Asterisks indicate tubules containing GATA1⁺ Sertoli cells. (E and F) Rac1 cKO testes (F) express AMH at similar levels to controls (E). Asterisks indicate tubules in (F). (G and H) Rac1 cKO tubules (H) show robust expression of GDNF within Sertoli cells, similar to controls (G). (I and J) Relative to controls (I), cKO testes (J) show abnormal cytoplasmic localization of AR in Sertoli cells (white arrowheads), but normal nuclear localization of AR in peritubular myoid cells (black arrowheads) and Leydig cells (white arrows). Thin scale bar, 100 μm; thick scale bar, 25 μm. (K) Average testis weight of P90 control (Rac1^flox/flox) males versus cKO males (n = 3 testes for controls and n = 2 testes for cKO, each from independent males). (L and M) (L) Average tubule cross-sectional area of P90 control (Dhh-Cre;Rac1^flox/+) versus cKO tubules (n = 10 tubules each from 3 independent males). (M) Cell counts of SOX9⁺ cells per tubule in P90 control (Dhh-Cre;Rac1^flox/+) versus cKO testes (n = 10 tubules each from 3 independent males). The data in (K)–(M) are shown as means ± SDs. p values were performed via a two-tailed Student’s t test. See also Figures S1–S3.

Figure 2.. Cell-Cycle Status and Onset of Sertoli Cell Quiescence Is Normal in Rac1 cKO Testes

(A–L) Three-month-old (P90) (A–D, I–L), P18 (E and F), and P24 (G and H) control Dhh-Cre;Rac1^flox/+ (A, C, E, G, I, and K) and Dhh-Cre;Rac1^flox/flox cKO (B, D, F, H, J, and L) testes. (A)–(D’), (K’), and (L’) are higher-magnification images of the boxed regions in (A)–(D), (K), and (L). (A and B) Both P90 control (A) and cKO (B) GATA1⁺ Sertoli cells are negative for MKI67. (C and D) While vimentin⁺ Sertoli cells are MKI67⁻ (arrowheads), TRA98⁺ germ cells are MKI67⁺ (arrows) in both control (C) and cKO (D) testes. (E–H) Both control (E and G) and cKO (F and H) Sertoli cells are MKI67 at P18 (E and F) and P24 (G and H). (I and J) Relative to controls (I), cKO testes (J) show an increase in cell death within tubules. (K and L) While virtually all of the apoptotic cells in control testes (K) are TRA98⁺ germ cells (arrowheads), some vimentin⁺ Sertoli cells are apoptotic in cKO (L) tubules (arrow in L). Dashed outlines indicate tubule boundaries. Thin scale bar, 100 μm; thick scale bar, 25 μm. (M) Average percentage of tubules containing cleaved caspase 3⁺ cells or cleaved caspase 3/TRA98⁺⁺ cells in P90 control versus cKO testes (n = 50 tubules each from 3 independent males). The data are represented as means ± SDs. p values were performed via a two-tailed Student’s t test.

Figure 3.. Rac1 Function in Sertoli Cells Is Required for Later Stages of Adult Spermatogenesis

(A–J) Three-month-old (P90) control (Dhh-Cre;Rac1^flox/+) (A, C, E, G, and I) and Dhh-Cre;Rac1^flox/flox cKO (B, D, F, H, and J) testes. (A’), (B’), (E’), (F’), (I’), and (J’) are higher-magnification images of the boxed regions in (A), (B), (E), (F), (I), and (J). Dashed outlines indicate tubule boundaries. (A and B) GFRA1⁺ undifferentiated spermatogonia (arrowheads) are at a basal location in both control (A) and cKO (B) tubules, while cKO Sertoli cells are often observed in the center of tubules (arrows in B’). (C–F) ZBTB16-bright/STRA8 undifferentiated spermatogonia (white arrowheads in E’ and F’), ZBTB16-dim/STRA8-dim differentiating spermatogonia (black arrowheads in E’ and F’), and ZBTB16/STRA8-bright spermatocytes (arrows in E’ and F’) are present in both control (C and E) and cKO (D and F) testes. (G and H) H1T⁺ meiotic and post-meiotic cells are visible in cKO testes (H), although in reduced numbers relative to controls (G). (I and J) γH2AX/H1T⁺⁺ spermatocytes with XY-body-enriched γH2AX staining (arrows) and H1T-bright round spermatids (arrowheads) are observed in both control (I) and cKO (J) tubules. Thin scale bar, 100 μm; thick scale bar, 10 μm. (K) Average number of TRA98⁺ germ cells per tubule in P90 control versus cKO (n = 3 tubules each from 3 independent control males and n = 15 tubules each from 3 independent cKO males). (L) Average number of GFRA1⁺ cells per tubule in germ cell-containing tubules in P90 control versus cKO testes (n = 25 tubules each from 3 independent males). (M) Average percentage of tubules displaying SOX9⁺ Sertoli cells mislocalized within the tubule lumen in P90 control versus cKO testes (n = 20 tubules each from 3 independent control males and n = 50 tubules each from 3 independent cKO males). (N) Quantitative real-time PCR analyses showing reduction in germ cell gene expression in P90 cKO testes relative to controls (n = 3 testes, each from independent males). The data in (K)–(N) are shown as means ± SDs. p values were performed via a two-tailed Student’s t test.

Figure 4.. Apicobasal Cell Polarity and BTB Are Disrupted in Adult Rac1 cKO Sertoli Cells

(A–N) Three-month-old (P90) (A)–(L) and 1-month-old (P30) (M and N) control Dhh-Cre;Rac1^flox/+ (A, C, E, G, I, K, and M) and Dhh-Cre;Rac1^flox/flox cKO (B, D, F, H, J, L, and N) testes. (E’)–(N’) are higher-magnification images of the boxed regions in (E)–(N). Dashed outlines indicate tubule boundaries. (A and B) GATA1⁺/SOX9⁺ Sertoli cell nuclei (arrows) in controls (A) are localized basally, but cKO nuclei (B) are both basally and centrally localized within the tubule. (C and D) Relative to controls (C), Vimentin is localized ectopically throughout the entire cKO Sertoli cell (D). (E and F) SCRIB is localized to the BTB and basal Sertoli compartment in both control (E) and cKO (F) testes. However, phalloidin staining is localized ectopically throughout the entire cKO Sertoli cell (see also I–L). (G and H) CLDN11 and CTNNB1 are enriched in a specific ring-like network (BTB; arrows in G^’) in control Sertoli cells (G), but they are localized throughout the entire cKO Sertoli cell (H). (I and J) PARD3 is normally enriched at the BTB and apical ES in controls (I), but it is localized throughout the entire cKO Sertoli cell (J). Note that PARD3 is also localized to the BTB. (K and L) ITGB1 is localized diffusely throughout cKO tubules (L), in contrast to specific apical ES staining in controls (K); however, basement membrane, spermatogonial, and interstitial staining is still observed in both control and cKO testes. (M and N) One-month-old (P30) control (Dhh-Cre;Rac1^flox/+) (M) and cKO (N) testes injected with biotin (and detected with streptavidin). cKO testes showed widespread infiltration of biotin deep into tubule lumens (arrows). Scale bar, 50 μm. See also Figure S4.

Figure 5.. Rac1 Function in Sertoli Cells Is Dispensable for the Maintenance of Undifferentiated Spermatogonia

(A–N) Seven-month-old control Dhh-Cre;Rac1^flox/+ (A, C, E, G, I, K, and M) and Dhh-Cre;Rac1^flox/flox cKO (B, D, F, H, J, L, N) testes. (C’), (D’), (K’), and (L’) are higher-magnification images of the boxed regions in (C), (D), (K), and (L). Dashed outlines indicate tubule boundaries. (A and B) Relative to controls (A), cKO testes (B) have smaller tubules, containing a variable number of TRA98⁺ germ cells. (C–H) Similar to controls (C, E, and G), cKO testes (D, F, and H) still have GFRA1⁺ and ZBTB16⁺ undifferentiated spermatogonia within tubules (arrows in C–F); cKO testes also contain a variable number of DDX4⁺ germ cells, albeit in reduced numbers. (I–L) As in controls (I and K), cKO testes (J and L) contain STRA8⁺ differentiating spermatogonia and spermatocytes (asterisks in I and J), as well as KIT⁺ differentiating spermatogonia (arrows in K’ and L’). (M and N) Relative to controls (M), cKO tubules (N) have a reduced, variable number of H1T⁺ spermatocytes and round spermatids. Thin scale bar, 100 μm; thick scale bar, 25 μm.

Figure 6.. Sertoli Rac1 Function Is Largely Dispensable for Postnatal Testicular Development

(A–N) P7 control Rac1^flox/flox (A, C, E, G, I, K, and M) and Dhh-Cre;Rac1^flox/flox cKO (B, D, F, H, J, L, and N) testes. (A’)–(F’) and (K’)–(N’) are higher-magnification images of the boxed regions in (A)–(F) and (K)–(N). (A–D) As in controls (A and C), P7 cKO testes (B and D) possess SOX9⁺ Sertoli cells, GFRA1⁺ undifferentiated spermatogonia, and DDX4⁺/KIT⁺ differentiating spermatogonia. Germ cells in cKO testes, similar to controls, have migrated to the tubule periphery. (E–H) Compared to controls (E and G), P7 cKO testes (F and H) express slightly lower levels of AMH, less nuclear AR in Sertoli cells (although interstitial expression and nuclear localization are normal), and slightly lower levels of GATA1. (I and J) Relative to controls (I), a significant increase in apoptosis is observed in cKO tubules (J). (K and L) Apoptosis is observed in mostly TRA98⁺ germ cells (arrowheads) in controls (K), but it is also observed in Sertoli cells (arrows) in cKO testes (L). (M and N) Most Sertoli and germ cells in both controls (M) and cKO tubules (N) are MKI67⁺. Thin scale bar, 100 μm; thick scale bar, 25 μm. (O) Quantitative real-time PCR analyses reveal no difference in germ cell gene expression in P7 control versus cKO testes (Ddx4, Pou5f1, Gfra1, Stra8, and Id4), but they do reveal a significant reduction in the Sertoli-expressed genes Sox9 and Ar, with no significant change in Amh and Gata1 expression (n = 4–6 testes for controls and cKO, each from independent males). (P) Graph showing slightly reduced Sertoli cell numbers in P7 cKO testes, but no loss of either DDX4⁺ or GFRA1⁺ germ cells (n = 10 tubules each from 3 testes, each testis from independent males). (Q) Average tubule cross-sectional area of P7 control versus cKO testes (n = 10 tubules each from 3 independent males). (R) Average number of tubules with CASP3⁺ cells and CASP3⁺/TRA98⁺ germ cells in P7 control versus cKO testes (n = 50 tubules each from 3 independent males). The data in (O)–(R) are shown as means ± SDs. p values were calculated using a two-tailed Student’s t test. See also Figures S5 and S6.

Figure 7.. Sertoli Rac1 Function Is Dispensable for Fetal Testis Differentiation

(A–L) E18.5 Rac1^flox/flox control (A, C, E, G, I, and K) and Dhh-Cre;Rac1^flox/flox cKO (B, D, F, H, J, and L) fetal testes. (A’), (B’), (K’), and (L’) are higher-magnification images of the boxed regions in (A), (B), (K), and (L). Dashed lines indicate tubule boundaries. (A and B) Both control (A) and cKO (B) tubules exhibit normal morphology and presence of SOX9⁺ Sertoli cells. CD45⁺ immune cells are observed within tubules in cKO testes (arrows in B and B’). (C and D) Both control (C) and cKO (D) testes show normal vascularization (PECAM1⁺ cells) and Leydig cell development (CYP17A1⁺ cells). (E and F) Relative to controls (E), cKO testes (F) exhibit an increase in apoptosis (cleaved caspase 3⁺ cells). (G–J) Both control (G and I) and cKO (H and J) tubules contain MKI67⁺ Sertoli cells (AMH⁺) and MKI67 germ cells (TRA98⁺). (K and L) Control (K) and cKO (L) tubules show similar expression of CTNNB1. Thin scale bar, 100 μm; thick scale bar, 25 μm. (M) Cell counts of SOX9⁺ cells per tubule in control versus cKO E18.5 testes (n = 15 tubules each from 3 independent males). (N) Average percentage of tubules containing cleaved caspase 3⁺ cells or cleaved caspase 3/TRA98⁺⁺ cells in control versus cKO E18.5 testes (n = 50 tubules each from 3 independent males). The data in (M) and (N) are shown as means ± SDs. p values were calculated using a two-tailed Student’s t test. See also Figure S7.