Retinoblastoma protein plays multiple essential roles in the terminal differentiation of Sertoli cells

Abstract

Retinoblastoma protein (RB) plays crucial roles in cell cycle control and cellular differentiation. Specifically, RB impairs the G(1) to S phase transition by acting as a repressor of the E2F family of transcriptional activators while also contributing towards terminal differentiation by modulating the activity of tissue-specific transcription factors. To examine the role of RB in Sertoli cells, the androgen-dependent somatic support cell of the testis, we created a Sertoli cell-specific conditional knockout of Rb. Initially, loss of RB has no gross effect on Sertoli cell function because the mice are fertile with normal testis weights at 6 wk of age. However, by 10-14 wk of age, mutant mice demonstrate severe Sertoli cell dysfunction and infertility. We show that mutant mature Sertoli cells continue cycling with defective regulation of multiple E2F1- and androgen-regulated genes and concurrent activation of apoptotic and p53-regulated genes. The most striking defects in mature Sertoli cell function are increased permeability of the blood-testis barrier, impaired tissue remodeling, and defective germ cell-Sertoli cell interactions. Our results demonstrate that RB is essential for proper terminal differentiation of Sertoli cells.

Figure 1

Deletion of RB in mutant testes occurs specifically in Sertoli cells. A, PCR amplification of DNA extracted from whole testis of Rb^flox/− mice of 4, 6, and 8 wk of age. Primers flanking the loxP sites amplify the recombined Rb conditional allele producing a 260-bp product. B, Quantitative RT-PCR of RNA extracted from isolated Sertoli cells of 6-wk-old animals. Primers were designed flanking the exon 19–20 boundary to show recombination of the Rb conditional allele (**, P < 0.01; control, n = 6; Rb cKO, n = 5). Immunohistochemical staining of RB in 6-wk-old control (C) vs. Rb cKO (D) testis in which Rb staining is ablated in Sertoli cells (Sc) but persistent in spermatogonia (Sg). RQ, Relative quantification.

Figure 2

Sertoli cell-specific Rb ablation causes progressive infertility. The fertility of control mice (n = 6) was compared with Rb cKO mice (n = 9) over 6 months. Comparisons of litter size per month per mouse (A) and pups per litter per mouse (B) show the progressive nature of the infertility. **, P < 0.01.

Figure 3

Progressive testicular atrophy and seminiferous tubule breakdown occurs in Rb cKO mice. Comparisons of body weights (A) and testis weights (B) from 5–11 wk of age show that although body weights do not vary significantly between groups, Rb cKO testis weights significantly decrease at 7 wk of age (P < 0.05) and progressively decrease to less than 19% of the control weights by 11 wk of age (n = 6–10 per data point). Gross morphology (C–F) and periodic Schiff (PAS) staining (G–N) of control (C, G, and K) compared with Rb cKO (D–F, H–J, and L–N) testes. A full complement of germ cells can be observed in control (G and K) and Rb cKO (H and L) testes at 6 wk of age, but representative sections from 8 (I and M) and 10-wk-old (J and N) testes show progressive loss of maturing germ cells in addition to reduction of tubular and luminal widths and increased vacuolization. Asterisks (I) indicate tubules in the 8-wk-old Rb cKO testis that had lost elongating spermatids and spermatozoa. Progressive germ cell loss is presumably not completely caused by loss of Sertoli cells because their presence can be identified in these dysfunctional tubules (arrowheads, M and N). C–F, 2 mm; G–J, 100 μm; K–N, 20 μm.

Figure 4

Rb cKO Sertoli cells have abnormal expression of markers of maturity. A, Although the testes of 8-wk old Rb cKO mice are smaller than those of the control, the other androgen-dependant organs of the Rb cKO urogenital system appear unchanged (sv, seminal vesicle; vd, vas deferens; e, epididymis; t, testis). B, Western blot analysis of the Sertoli cell markers AR and SOX9 in the control (lane 1) and Rb cKO (lane 2) 6-wk-old whole testis lysates show comparable expression whereas p27 is decreased significantly in Rb cKO [*, P < 0.05, comparing band densitometry between groups (n = 3,3) normalized to β-tubulin (β-TUB)]. C–F, Changes in Sertoli cell function are seen by comparing AR immunohistochemistry in 8- (E) and 10-wk-old (F) Rb cKO testes to 6-wk-old control (C) and Rb cKO (D) testes. Arrows (E) indicate sloughed Sertoli cells present in the tubular lumen of 8-wk-old Rb cKO mice. Arrowheads (F) indicate misregulated expression of AR in 10-wk-old Rb cKO Sertoli cells showing variable intensities of staining. Cont, Control.

Figure 5

RB deletion causes impairment of Sertoli cell cycle quiescence. A, Venn diagram of microarray changes in E2F-related, cell cycle-regulatory, and DNA synthesis genes shows the overlap between these pathways. B, Quantitative RT-PCR of select (bold) genes from panel A, and E2f1 confirms the up-regulation of these genes in Rb cKO Sertoli cells (*, P < 0.05; **, P < 0.01; control, n = 6; Rb cKO, n = 5). RQ, Relative quantification. C–F, Immunofluorescent analysis of BrdU (red) and Sox9-GFP (green) indicates that occasional 6-wk-old Rb cKO Sertoli cells show persistent DNA synthesis (S phase). Arrows (C) indicate Sertoli cells that were not BrdU positive.

Figure 6

RB deletion in Sertoli cells causes activation of apoptotic pathways. A, Venn diagram of microarray changes in E2F-related, p53-related, and apoptotic genes shows the overlap between these pathways. B, Quantitative RT-PCR of select (bold) genes from panel A, and Trp53 confirms the up-regulation of these genes in Rb cKO Sertoli cells (*, P < 0.05; **, P < 0.01; control, n = 6; Rb cKO, n = 5). RQ, Relative quantification. C–E, TUNEL (green) staining in 6-wk-old testes is increased in Rb cKO (D) as compared with control (C) as determined by apoptotic index (E) (**, P < 0.01; control, n = 3; Rb cKO, n = 4). F–I, Immunofluorescent analysis of cleaved caspase-3 (red) and Sox9-GFP (green) reveals the activation of caspase-mediated apoptosis in 6-wk-old Rb cKO Sertoli cells. Arrows (I) indicate Sertoli cells that did not stain for cleaved caspase-3. DAPI, 4′,6-diamidino-2-phenylindole.

Figure 7

Infertility in Rb cKO mice may be caused by abnormal expression of genes important to tight junction formation and tissue remodeling. A, Venn diagram of changes in AR-related, cell adhesion molecules (CAMs) and tight junctions (TJs), and proteases and their inhibitors shows the overlap between these pathways. B, Quantitative RT-PCR of select (bold) genes from panel A and Ar confirms the misregulation of these genes in Rb cKO Sertoli cells (*, P < 0.05; **, P < 0.01; control, n = 6; Rb cKO, n = 5). RQ, Relative quantification. C and E, A biotin tracer (red) was injected into 6-wk-old control (C) and Rb cKO (E) testes. Presence of biotin in the adluminal space of the Rb cKO mice indicates that these mice have a defect in blood-testis barrier function. C–F, Espin (green) marks the basal ectoplasmic specializations (white arrows) of the blood-testis barrier. The normal curvilinear staining of the basal ectoplasmic specializations in the control (D) has been lost in Rb cKO (F). The insets show the proximity between the blood-testis barriers and the basement membranes (dashed line). Espin also marks the apical ectoplasmic specializations (red arrows) that are important for formation of the sperm head, and this staining is neither organized nor polarized in Rb cKO (F) as they are in the control (D).

Figure 8

A summary of the postulated role of RB in Sertoli cells. In wild-type (WT) Sertoli cells, inhibition of E2F3a by RB prevents expression of E2F1. In Rb cKO Sertoli cells, the aberrant activation of E2F3a and, thus, E2F1 leads to derepression of cell cycle and apoptotic genes. RB may also modulate the expression of AR-regulated genes in WT Sertoli cells; therefore, the lack of RB in Rb cKO Sertoli cells may directly lead to their misregulation. Repression of E2F3a by RB may be responsible for AR-independent gene changes involved in adult Sertoli cell function (i.e. Timp1 down-regulation).