Partially redundant functions of SRC-1 and TIF2 in postnatal survival and male reproduction

Abstract

Both SRC-1 and TIF2 are members of the p160 steroid receptor coactivator family. Genetic analyses have shown that inactivation of TIF2, but not SRC-1, reduces postnatal survival, growth, and male reproductive function. Here, we demonstrate that, through analyses of SRC-1/TIF2 compound mutant mice, SRC-1 can partially compensate for the effects of a loss of TIF2 on mouse survival and growth, whereas SRC-1 and TIF2 are dispensable for primary organogenesis. The highly variable onset of defects observed in TIF2(-/-) testes due to the absence of TIF2 in Sertoli cells, including abnormal spermiogenesis, age-dependent degeneration of seminiferous epithelium, and disorder of cholesterol homeostasis, is uniformly accelerated upon inactivation of SRC-1 alleles in the TIF2 null genetic background, thus demonstrating that TIF2 and SRC-1 can perform redundant functions in Sertoli cells. Massive desquamation of immature germ cells together with an increase in germ cell apoptosis and a decrease in germ cell proliferation may be responsible for the early onset of the severe seminiferous epithelial degeneration observed in SRC-1(+/-)/TIF2(-/-) testes. Interestingly, the overall abnormal features displayed by the SRC-1(+/-)/TIF2(-/-) and SRC-1(-/-)/TIF2(-/-) mutant testes, including spermatid maturation defects, increase in Sertoli cell lipid stores, loss of immature germ cells, and formation of giant multinucleated spermatids, are commonly detected in testes of elderly men, suggesting that deficiencies in molecular pathways involving TIF2 and SRC-1 in Sertoli cells could participate in testicular senescence.

Fig. 1.

Failure of lung alveoli expansion (a and b) at birth and reproductive defects (c–m) in SRC-1/TIF2 compound mutants. (a and b) Histological sections from E18.5 WT (a) and SRC-1^–/–/TIF2^–/– (b) lungs: 30 min after cesarian delivery, mutant alveoli are still collapsed. (c–m) Histological sections of epididymides (c–e) and testes (f–m) of 9-week-old WT, TIF2^–/–, and SRC-1^+/–/TIF2^–/– males. Oligozoospermia and testicular degeneration are observed only in compound mutants. Mild forms of the spermiation failure and Sertoli cell lipid accumulation, which are consistently observed in compound mutants are occasionally seen in their TIF2^–/– littermates. A, alveoli; B, bronchi; L, Leydig cells; P, pachytene spermatocytes; R, retained, mature spermatids; RS, round spermatids; S, Sertoli cells; St9, elongating, step 9, spermatids; SY, symplasts; T, seminiferous tubules; T*, Sertoli cell-only seminiferous tubule; V, vacuole; Z, spermatozoa. IX, stage nine of the seminiferous epithelium cycle. The arrowheads point to lipid droplets which are blackened by osmium fixation of the tissues. Shown are Mallory's trichrome stain (a and b) and osmium tetroxide fixation with (c–g and k–m) or without (h–j) toluidine blue stain. (Bars are 100 μmin a and b, 10 μmin c–e and h–m, and 30 μmin f and g.)

Fig. 2.

Elongating spermatids are abnormal in SRC-1^+/–/TIF2^–/– males. Analysis by electron microscopy of WT (a and c) and SRC-1^+/–/TIF2^–/– (b, d, and e) step 12 spermatids at 9 weeks of age. A, acrosome; M, manchette; MI, mitochondria; MS, spermatocyte in metaphase; N, nucleus. (Bars are 2 μmin a and b and 0.6 μmin c–e.)

Fig. 3.

Increased apoptosis and decreased germ cell proliferation in testes from young SRC-1^+/–/TIF2^–/– males. (a) Analysis of apoptotic cell death at 9 weeks of age. TUNEL-positive cells were counted on 30 seminiferous tubule cross sections from five mice. Data are presented as the average number of TUNEL-positive cells per cross section. **, P < 0.01 vs. the other two groups in unpaired t test. (b) Analysis of cell proliferation by immunohistochemical detection of PCNA at 5 (black bars) and 9 (white bars) weeks of age. PCNA-positive and -negative cells in contact with basement membranes of the seminiferous tubules (which include spermatogonia and early-stage spermatocytes) were counted were counted on 10 tubule cross sections from four mice, and data are presented as mean percentage of PCNA-positive cells. **, P < 0.01 vs. the other two groups in unpaired t test. (c and d) Histological sections of seminiferous tubules of 9-week-old mice; d provides an example of abnormal tubular cross section displaying a normal amount of round spermatids but lacking almost all spermatogonia and spermatocytes. P, pachytene spermatocytes; PR, preleptotene spermatocytes; RS, round spermatids; S, Sertoli cells; SG, spermatogonia. A hematoxylin and eosin stain was used. (Bar is 40 μm.)

Fig. 4.

Immunohistochemical detection of SRC-1 in the seminiferous epithelium. Histological sections from SRC-1^–/– (a) and WT (b) testes were immunostained with antibodies to SRC-1. SRC-1 immunoreactivity in Sertoli cell nuclei (arrows) was detected in WT, but not in SRC-1^–/– mice. Sections were counterstained with methylgreen. (Bar is 50 μm.)