Germ Cell-Specific Retinoic Acid Receptor α Functions in Germ Cell Organization, Meiotic Integrity, and Spermatogonia

Abstract

Retinoic acid receptor α (RARA), a retinoic acid-dependent transcription factor, is expressed in both somatic and germ cells of the testis. Rara-null male mice with global Rara mutations displayed severely degenerated testis and infertility phenotypes. To elucidate the specific responsibility of germ cell RARA in spermatogenesis, Rara was deleted in germ cells, generating germ cell-specific Rara conditional knockout (cKO) mice. These Rara cKO animals exhibited phenotypes of quantitatively reduced epididymal sperm counts and disorganized germ cell layers in the seminiferous tubules, which worsened with aging. Abnormal tubules lacked lumen, contained vacuoles, and showed massive germ cell sloughing, all characteristics similar to those observed in Rara-null tubules. Spermatocyte chromosomal spreads revealed a novel role for germ cell RARA in modulating the integrity of synaptonemal complexes and meiotic progression. Furthermore, the initiation of spermatogenesis from spermatogonial stem cells was decreased in Rara cKO testes following busulfan treatment, supporting a role of germ cell RARA in spermatogonial proliferation. Collectively, the evidence in this study indicates that RARA produced in male germ cells has a broad spectrum of functions throughout spermatogenesis, which includes the maintenance of seminiferous epithelium organization, the integrity of the meiotic genome, and spermatogonial proliferation and differentiation. The results further suggest that germ cell RARA has dual functions: intrinsically in germ cells, balancing proliferation and differentiation of spermatogonia, and controlling genome integrity during meiosis; and extrinsically in the crosstalks with Sertoli cells, controlling the cell junctional physiology for coordinating proper spatial and temporal development of germ cells during spermatogenesis.

Figure 1.

Floxed Rara construct and breeding scheme for germ cell–specific excision of exon 4, coding for a portion of the B domain on RARA. (a) Rara targeting vector with black triangles indicating locations of loxP sites flanking the region containing exon 4 (E4, “floxed” region). Numeric notations indicate base pairs with internal reference to gene coding sequence, and alphanumeric notations indicate locations of restriction enzyme activities. (b) Two-step breeding scheme to obtain homozygous Rara germ cell–specific knockout (Rara cKO: Stra8^iCre/+;Rara^−/−) animals using Rara floxed females (left) and Stra8-iCre males (right). Numeric and alphanumeric notations with arrows beneath each construct indicate primers (Table 1) and primer directions used in genotyping. B, BamHI; C, ClaI; E, EcoRI; N, NotI; Nd, NdeI; Neo, neomycin; Nh, NheI; polyA, polyadenylation; SD/SA, splice donor/splice acceptor; SV40, simian virus 40; TK, thymidine kinase; X, XhoI.

Figure 2.

Verification of Rara inactivation in germ cells. (a) Mating between the R26R-Eyfp females carrying an iCRE-activity reporter construct and Stra8-iCre males leads to EYFP expression in germ cells. CCAG, chicken β-actin promoter. (b–e) Green fluorescent cells are germ cells with iCRE activity (white arrowheads) in testis cross-sections from animals at P1, P3, P5, and P7. (f) Gonocytes with iCRE activity are EYFP- and ZBTB16-positive germ cells (white pointed arrowheads) in P1 testis cross-sections. Scale bars, 50 µm. (g) Percentage of tubules containing at least one EYFP-positive cell in animals at P1, P3, P5, and P7. n = 3 to 4 for P1, P3, and P7; n = 2 for P5. Error bars are SD. (h and i) IHC for RARA on testicular sections from WT and Rara cKO mice at P8. Black arrowheads indicate Sertoli cells; black arrows indicate germ cells. n = 3. Scale bars, 50 µm. (j) PCR products obtained using DNA template isolated from epididymal sperm heads in WT and cKO animals at P75. A 487-bp fragment represents the deleted form of Rara allele, and a 554-bp fragment represents the WT Rara allele. n = 3 to 5. (k) Real-time RT-qPCR analysis on RNA collected from enriched germ cell populations from WT (open bar) and Rara cKO (cross-hatched bar) animals at P4 and P8 for Rara (***P < 0.001; WT and cKO n = 3) and Stra8 (**P < 0.01, ***P < 0.001; WT n = 6, cKO n = 4). Error bars are SEM. L, DNA ladder.

Figure 3.

Histological analyses show increased morphological abnormalities in testicular and epididymal cross-sections of Rara cKO animals. Hematoxylin and eosin–stained testis cross-sections of (a, c, e, g, i, k, m, o, and q) WT and (b, d, f, h, j, l, n, p, and r) Rara cKO animals at various ages from P15 to P365. (a, c, and g) Black arrowheads in WT animals show most advanced cell types at P15, P20, and P30, which are pachytene spermatocytes, round spermatids, and elongated spermatids, respectively. (s and t) Hematoxylin and eosin–stained epididymis cross-sections of (s) WT and (t) Rara cKO mice at P75. Insets in (t) show immature cells at a higher magnification. Seminiferous tubules in Rara cKO animals showed (f, j, and l) sloughing cells blocking the lumen, vacuolization, and (n, p, and r) missing germ cell layers. Scale bars, 50 µm in (a)–(r). Scale bars, 100 µm in (s) and (t).

Figure 4.

Quantitation of increased seminiferous tubule abnormalities, delayed progression of germ cell development, decreased sperm count and testis weights, and compromised BTB in Rara cKO testes. (a) Percentage of tubules with abnormalities was present in Rara cKO mice (cross-hatched bar) compared with WT mice (open bar) at P15 and onward, up to P365 (*P < 0.05, **P < 0.01). n = 5 to 10. Error bars are SEM. (b) Tubules containing sloughed germ cells were quantified in mice at P45, P75, P120, and P180, presented as fold change relative to the WT level (*P < 0.05, **P < 0.01). n = 4 to 5. Error bars are SEM. (c) Percentage of tubules containing the most advanced cell types in the testis of Rara cKO mice compared with WT mice at P15, P20, and P30 (***P < 0.001). n = 3. Error bars are SEM. (d) Sperm counts, shown in millions per milliliter, in mice at P75, P120, P180, and P365 (**P < 0.01, ***P < 0.001). n = 5 to 14. Error bars are SEM. (e) Testis weights, measured in milligrams (*P < 0.05, **P < 0.01). n = 9 to 29. Error bars are SEM. (f–h) Tubules were scored for the presence of biotin in the adluminal compartment (red), presented as fold change relative to the WT level (*P < 0.05). n = 3 to 4. Error bars are SEM. Scale bars, 50 µm.

Figure 5.

Meiotic progression delayed in Rara cKO mice. (a–d) Testicular cross-sections of WT and Rara cKO mice at P11 and P15; γH2AX-positive cells are in red. L/Z indicates tubules containing leptotene and zygotene spermatocytes; P indicates tubules containing pachytene spermatocytes. Scale bars, 100 µm. (e) Quantification of the percentage of tubules with γH2AX-positive leptotene and zygotene spermatocytes for WT and Rara cKO animals at P11, P15, and P20 ages (*P < 0.05, **P < 0.01). n = 3 to 8. Error bars are SEM. (f and g) From chromosomal spreads, the percentages of meiotic prophase cells in leptotene/zygotene and pachytene stages are shown for WT (blue circles) and Rara cKO (orange squares) mice at P15, P17, P19, and P21 ages (*P < 0.05). n = 3 to 5. Error bars are SEM.

Figure 6.

Defective synapsis and increased SC fragmentation in pachytene spermatocytes, followed by increased apoptosis in Rara cKO mice. (a–c and e–g) Chromosomal spreads of pachytene spermatocytes from WT and Rara cKO animals at P15. SYCP3 immunostain showing SC (red), γH2AX immunostain showing DNA double-stranded breaks (green), and DAPI DNA stain (blue) were used. White box in (a) outlines perfect synapsis enlarged in (e). White arrows in (b) and (c) indicate chromosomal fragmentation; a representative outlined by a white box in (b) is enlarged in (f). White arrowheads in (b) and (c) indicate autosomal γH2AX staining; a representative outlined by a white box in (c) is enlarged in (g). Scale bars, 10 μm in (a–c); 2.5 µm in (e–g). (d) From chromosomal spreads, the percentage of cells with perfect synapsis (gray), containing fragmented SC segments (white), and containing other defects (forks, gaps, and partial asynapsis) (black) are shown (^‡P < 0.1, *P < 0.05). n = 3 to 5. (h–k) Apoptotic cells visualized by TUNEL assay (green) and DNA visualized by DAPI stain (blue) in seminiferous tubules from WT and Rara cKO mice at P17 and P19. Scale bars, 100µm. (l) Percentage of tubules containing three or more apoptotic cells (^‡P < 0.1, *P < 0.05). n = 5 to 6. Error bars are SEM.

Figure 7.

Decreased regenerative capacity of SSCs. (a–d) Hematoxylin and eosin–stained testis cross-sections of (a) WT and (b) Rara cKO animals injected with busulfan and allowed to recover for (a and b) 4 wk or (c and d) 8 wk. Scale bars, 100 µm. (e) Quantification of germ cell regeneration in WT and Rara cKO animals at 4- and 8-wk recovery times (^‡P < 0.1, *P < 0.05). n = 6 to 11. Error bars are SEM. E, empty tubule; R, regenerated tubule.

Figure 8.

Schematic drawing showing three potential functions of germ cell RARA evident from the characterization of germ cell–specific Rara cKO mouse model. (a) Germ cell RARA has a direct role in meiotic prophase spermatocytes (dark purple), keeping SC integrity. (b) Preleptotene spermatocyte (PL; light purple) expresses RARA at the highest level in the testis. We propose that germ cell RARA in PL regulates crosstalk (double-ended arrows) with Sertoli cells (yellow) for PL to traverse BTB. PL breaks the inter–Sertoli tight junction (TJ1; blue) and enters into the transient compartment where the TJ2 (blue) ahead is still intact. In the transient compartment, germ cells interact with Sertoli cells via junctional molecules (orange ovals), which also mediate Sertoli–germ cell contact in the adluminal compartment. When TJ1 reforms behind PL, PL breaks TJ2 to enter into the adluminal compartment, past the BTB. (c) Germ cell RARA has a function in progenitor spermatogonia (PS, SSC; light green), similar to germ cell RARG, for the spermatogonial differentiation, producing differentiated spermatogonia (DS; dark green) and the initiation of meiosis. White arrows throughout indicate germ cell differentiation steps.