The steroid receptor coactivator SRC-3 (p/CIP/RAC3/AIB1/ACTR/TRAM-1) is required for normal growth, puberty, female reproductive function, and mammary gland development

Abstract

Steroid receptor coactivator-3 (SRC-3) is a coactivator of nuclear receptors in the SRC family as assayed in vitro. Here, we show that mouse SRC-3 is expressed in a tissue-specific fashion and distributed mainly in the oocytes, mammary glands, hippocampus, olfactory bulb, smooth muscle, hepatocytes, and vaginal epithelium. Genetic disruption of SRC-3 in mice results in a pleiotropic phenotype showing dwarfism, delayed puberty, reduced female reproductive function, and blunted mammary gland development. Hormonal analysis indicates that SRC-3 plays a role in both the growth hormone regulatory pathway and the production of estrogen, which may explain the observed phenotypes. These results suggest that the physiological role of SRC-3 is different from that of SRC-1 and prove the diversity among coactivator family members.

Figure 1

Generation of SRC-3^−/− mice. (A) Gene targeting strategies. Mouse SRC-3 gene is diagrammed to show its exons (black boxes), restriction enzyme sites, and codons for translational start (ATG) and stop (∗). Components of the targeting vector and the corresponding locations of its 5′ and 3′ homologous targeting arms in the gene are diagrammed. Bm, BamHI; Sm, SmaI; Hd, HindIII; Kp, KpnI; Rv, EcoRv. (B) Identification of SRC-3 mutant mice. Tail DNA was digested with BamHI and EcoRV. Southern blotting was performed with the 5′ probe (Upper). The 6-kb band for wild-type allele (wt) and the 7.2-kb band for the targeted allele (ko) are indicated. In PCR analysis (Lower), primer pairs P76/P77 and P76/P78 (see A for locations) were used to detect the wild-type allele (0.45 kb) and the targeted allele (0.23 kb), respectively. (C) Absence of SRC-3 protein in SRC-3^−/− mice. Western blot analysis was carried out by using tissue lysates prepared from mammary gland biopsy on lactation day 15 as described previously (21). The blot was probed with affinity-purified polyclonal Abs against human SRC-3 (37). A nonspecific (NS) band appears in all lanes and serves as a control for total amounts of protein loaded.

Figure 2

Tissue-specific expression of SRC-3. Sections of frozen tissues from 4-wk-old SRC-3^+/− mice were prepared for X-Gal staining (blue). (A) Ovarian section. O, oocytes; G, granulosa cells. (B) Cross section of the uterus. EM, uterine endometrium. (C) Whole mount of the mammary gland from a 6-wk-old SRC-3^+/− female. EB, end bud. (D) Coronal section of the hippocampus. DG, dentate gyrus. We note that occasionally ectopic expression of β-gal can be visualized in knock-in mice. Although Northern RNA analyses of tissues are in agreement with our patterns, this remains a caveat.

Figure 3

SRC-3^−/− mice exhibit growth retardation and low levels of IGF-1. (A and B) Growth curves for females and males with different SRC-3 genotypes. Body weight is presented as average ± SE. (C) Serum IGF-1 measurement. All mice were males with an average age of 39.3 ± 6.4 days. (D) Correlation between IGF-1 concentrations and body weight for SRC-3^−/− mice. Statistical t test indicates a very significant (P < 0.01) positive correlation (r = 0.828).

Figure 4

SRC-3^−/− females have delayed vaginal opening and low levels of E2. (A) Vaginal opening time. Individual distribution and average vaginal opening time are indicated for SRC-3^+/+, SRC-3^+/−, and SRC-3^−/− females. There are statistical differences (P < 0.001) between SRC-3^−/− and SRC-3^+/+/SRC-3^+/− mice. (B) Serum E2 concentrations in control and knockout females. Control mice include wild-type and SRC-3^+/− mice. The E2 values are presented as average ± SD (n = 8). E2 levels in knockout mice are statistically lower than those in control mice at all ages assayed (P < 0.01, t test).

Figure 5

E2 and P-stimulated alveolar formation is reduced in SRC-3^−/− mammary gland. Whole mount of mammary gland was prepared from E2 + P pellet-treated adult females with indicated SRC-3 genotypes. Note the dramatic difference in the amount of aborization and the number of alveoli (*).