Male sterility in mice lacking retinoic acid receptor alpha involves specific abnormalities in spermiogenesis

Abstract

The severe degeneration of the germinal epithelium and subsequent male sterility observed in mice null for the retinoic acid receptor alpha (RARalpha) gene suggested its critical role in spermatogenesis, although the etiology and progression of these abnormalities remain to be determined. Previous studies have revealed that elongated spermatids in RARalpha(-/-) testes were improperly aligned at the tubular lumen and did not undergo spermiation at stage VIII(*). We now report a distinctive failure of step 8-9 spermatids to orient properly with regard to the basal aspect of Sertoli cells, resulting in stage VIII(*)-IX(*) tubules with randomly oriented spermatids. By in situ terminal deoxynucleotidyltransferase-mediated deoxy-UTP nick end labeling (TUNEL), we noted that elongating spermatids frequently underwent apoptosis. Immunohistochemical analysis revealed that while activated caspase-3, the primary effector caspase in the apoptotic cell death machinery, was detected in the nuclei of primary spermatocytes in the first wave of spermatogenesis and occasionally in spermatogonia of both normal and mutant testes, it was not involved in the death of elongating spermatids in RARalpha(-/-) testes. Thus, sterility in RARalpha(-/-) males was associated with specific defects in spermiogenesis, which may correlate with a failure in both spermatid release and spermatid orientation to the basal aspect of Sertoli cells at stage VIII(*) in young adult RARalpha(-/-) testis. Further, the resulting apoptosis in elongating spermatids appears to involve pathways other than that mediated by activated caspase-3.

Fig. 1

Failure of spermatid release and aberrant orientation of spermatids of RARα^−/− testis at stage VIII within the seminiferous epithelium. The acrosome of spermatids in histological sections of testes from RARα^+/+(A, B, E) and RARα^−/− males (C, D, F) was detected with PAS staining (magenta color). For RARα^+/+ testis, early stage VIII (A), stage IX (B), stage IV (E) are shown next to corresponding stages in RARα^−/− testis (stage VIII*, C; stage IX*, D; stage IV*, F, respectively). Though the asynchronous cell associations within the seminiferous epithelium of the mutant testes make “staging” difficult, an attempt was made to stage the tubules using the acrosomal system (Russell et al., 1990). PS, pachytene spermatocytes; PL/L, preleptotene or leptotene spermatocytes; PL, preleptotene spermatocytes; L, leptotene spermatocytes; arabic numerals, the step of elongated spermatid. Roman numerals indicate the stage of the seminiferous tubule. A–D, × 100; E, F, × 60. Arrows in A–D indicate the orientation of elongated speramtids while the arrows in E and F show the entrenchment of spermatids (or lack thereof) within a recess of Sertoli cells. Arrowhead in A indicates step 16 spermatids.

Fig. 2

Comparison of histology and TUNEL-labeling in young adult testes of RARα^+/+ and RARα^−/− testis. Histological sections of 8- and 9-week-old (young adult) testes from RARα^+/+ (A) and RARα^−/− mice (B–E), with corresponding epididymis from RARα^+/+ (H) and RARα^−/− (I) males, are shown, along with histological sections of 6-week-old testis from RARα^−/− mice (F, G). Staining with hematoxylin revealed the appearance of TUNEL-positive spermatids at the basal lamina only in the young adult RARα^−/− testis (B, D, E) and not in the RARα^+/+ testis (A). Apoptotic elongated spermatids were detected when they were deeply embedded in the seminiferous epithelium of 6-week-old RARα^−/− testis (G, inset with high magnification). Panels A and B, × 20; C–E, × 60; H and I, × 60; F and G, × 40. Small panel in D shows magnification at × 100. Small panel in F–G, × 60. Arrowheads in F–G point to the TUNEL-positive spermatogenic cells. Arabic numerals in panel D indicate the step of elongated spermatid.

Fig. 3

Germ cell apoptosis during testicular development in RARα^+/+ and RARα^−/− testes. (A) A bar graph indicating the number of TUNEL-positive germ cells at various intervals after birth in RARα^+/+ and RARα^−/− mice (100 tubules scored per testis; three mice for each time point). The total number of apoptotic germ cells (excluding elongated spermatids) per 100 seminiferous tubules was counted in testicular sections of 2–9-week-old RARα^+/+ and RARα^−/− males. The bars represent the mean ± SD of three mice for each time point. (*) indicates significant differences within the same age group as well as between age groups by ANOVA, p<0.05. (**) indicates significant differences within the same age group as well as between age group by ANOVA, p<0.001. (***) indicates significant differences within the same age group as well as between age groups by ANOVA, p< 0.0001. (B) Typical round-shaped tubules with apoptotic heads of elongated spermatids at the periphery of the tubules are designated as T+. × 20. (C) Bar graph indicating the percentage of tubules with TUNEL-positive elongated spermatids at various intervals after birth in RARα^−/− mice (100 tubules scored per testis: three mice for each time point). Appearance of TUNEL-positive elongated spermatids was not observed in the tubules until they were 6 weeks of age. These unique TUNEL-positive elongated spermatids were never seen in RARα^+/+ testis at various intervals after birth (data not shown).

Fig. 4

Immunohistochemical localization of caspase-3 in juvenile and young adult testes of RARα^+/+ and RARα^−/− mice. Histological sections of testes from RARα^+/+ (A, B) and RARα^−/− males (C–H) were immunostained with anti-activated caspase-3 antibody and were assessed by TUNEL assay. A–H, × 40. P, primary spermatocytes. Left panel shows the section after TUNEL assay while the right panel shows the activated caspase-3-labeled cells in the next serial section of the corresponding age. Arrows in C and D point to the TUNEL-positive and activated caspase-3-labeled spermatogenic cells, respectively. Arrowheads in C point to the TUNEL-positive spermatogenic cells at the site of cells collectively undergoing apoptosis.

Fig. 5

Activated caspase-3 expression pattern during testicular development in RARα^+/+ and RARα^−/− testes. A bar graph indicating the number of activated caspase-3-positive germinal cells at various intervals after birth in RARα^+/+ and RARα^−/− mice (100 tubules scored per testis: three mice for each time point). The total number of activated caspase-3-labeled germ cells per 100 seminiferous tubules was counted in testicular sections of 2-4-, 6-, 8-week-old RARα^+/+ and RARα^−/− males. The bars represent the mean ± SD of three mice for each time point. (*) indicates significant differences within the same age group as well as between age groups by ANOVA, p<0.05. (**) indicates significant differences within the same age group as well as between age groups by ANOVA, p<0.01. (***) indicates significant differences within the same age group as well as between age groups by ANOVA, p<0.0001.