Fig 2: Pathological changes in the lungs and spleens of mice with aging. (A) Representative hematoxylin and eosin (HE)-stained images of aged wild-type (WT) and Secretoglobin (Scgb) 3a2-knockout (KO) mouse lungs. (i) Two-year-old WT mouse lungs exhibiting mild lymphocyte aggregation. (ii) Marked lymphocyte aggregation detected in 2-year-old KO mouse lungs. (iii) Atypical lymphocytes observed in 2-y-old KO mouse lung. Scale bars: 200 µm. (iv) Enlarged photo near the asterisk in c. Scale bar: 20 µm. (B) Macroscopic observations and diameters of spleens measured in senescent WT and Scgb3a2-KO mice. Representative spleens are presented. Scale bars: 5 mm. (C) Representative histological observations of spleen tissues from WT (i, iii, v, vii, x, and xiii) and Scgb3a2-KO mice (ii, iv, vi, viii, xi, and xiv) at various ages. In the spleen of 2-year-old KO mice, remarkable fusion of white pulp (xi) and a highly malignant image (ix: inset, ×1000) were observed. Asterisk (*): white pulp fusion; arrowheads: fiber formation; ix: enlargement of arrowheads in viii; xii: enlargement of * in xi; xiii: extramedullary hematopoiesis (erythroid element, megakaryocytes, and myeloid cells) in the splenic red pulp; xiv: erythropoiesis in red pulp. Scale bars: 200 µm (i–viii, x, and xi), 50 µm (xiii, xiv), and 10 µm (ix, xii).

Fig 3: Lm and DI in lung tissues during aging. (A) Mean linear intercept (Lm) in wild-type (WT) and Secretoglobin (Scgb) 3a2-knockout (KO) mouse lungs from 8 weeks (8 w) to 2 years of age (2 y). Lm of WT significantly increased in 2-year-old (2 y) in WT mice, whereas that in KO mice gradually increased after 1 year (1 y), being significantly larger than that in WT. (B) Destructive index (DI) in wild-type and Scgb3a2-KO mouse lungs from 8 w to 2 y. DI was constant regardless of age in WT mice, and after 1 year of age (1 y) in KO mice. Filled columns and black dots: WT; open columns and white dots: KO; N=5-9. Specifically, the number of mice analyzed were as follows: WT [8w: 3 males (m), 2 females (f); 0.5y: 4m, 4f; 1y: 3m, 2f; 1.5y: 5f; 2y: 3m, 5f] and KO (8w: 4m, 3f; 0.5y: 3m, 4f; 1y: 2m, 2f; 1.5y: 5m, 4f; 2y: 3m, 3f). Because Lm and DI did not apparently differ between male and female mice, the results were combined. Data are presented as the mean ± standard deviation (SD). Statistical differences between ages in the same strain were analyzed by one-way analysis of variance (ANOVA), followed by the Tukey–Kramer post hoc test (#p < 0.05, ##p < 0.01). Differences between different strains at the same age were analyzed using Student’s t-test (*p < 0.05, **p < 0.01).

Fig 4: Effects of SCGB3A2 stimulation on mRNA expression of anterior pituitary hormones in primary cultured rat pituitary cells. Rat anterior pituitary cells were cultured and stimulated by SCGB3A2 (300 ng/ml) for 24 h and mRNA were extracted. Expression of LH (a) and FSH (b) was quantified by real-time RT-PCR. Expression levels of LH and FSH were significantly inhibited by SCGB3A2 stimulation. *P < 0.01; n = 6–8

Fig 5: Double staining of SCGB3A2 and anterior pituitary hormones in the adult mouse pituitary gland. Immunohistochemistry for SCGB3A2 and growth hormone (a), prolactin (b), thyroid-stimulating hormone (c), luteinizing hormone (d), follicle-stimulating hormone (e) and adrenocorticotropic hormone (f). Brown signal indicates SCGB3A2, whereas blue signal indicates anterior pituitary hormones. Black arrowheads indicate immunopositive cells for anterior pituitary hormones and white arrowheads indicate double-positive cells for SCGB3A2 and anterior pituitary hormones. Bars 50 µm