Fig 2: IL-6 expression in epididymis of WT and Ncoa5+/− male mice. (A) Western blot analysis of IL-6 expression in epididymis of age-matched 13–15 months old WT (n = 5, lanes 1–5) and Ncoa5+/− (n = 6, lanes 6–11) male mice using anti-IL-6 and anti-β-actin antibodies. Each lane represents a single tissue lysate of the whole epididymis from a mouse. Cropped blots from the same gel are shown. Uncropped blots are presented in Supplementary Fig. S4. The same blot membrane was stripped and blotted with the same method except without adding the primary antibodies as control (Supplementary Fig. S4C). (B) Quantification of data from (A). n = the number of mice. **P < 0.01. Error bar ± SEM. (C) Representative IL-6 IHC staining of WT, Ncoa5+/− and Ncoa5+/−Il-6+/− male mouse epididymides. Immunohistochemical analysis was performed on epididymis from 4.5 months old WT (n = 2), Ncoa5+/− (n = 3) and Ncoa5+/−Il-6+/− (n = 3) male mice using an anti-IL-6 antibody. n = the number of mice. Representative IL-6 IHC staining images of WT (a–f), Ncoa5+/− (g–l) and Ncoa5+/−Il-6+/− (m–r) epididymal caput (segments 1–2) at lower (a, g and m) and higher (b, h and n) magnification, corpus (segments 7) at lower (c, i and o) and higher (d, j and p) magnification, and cauda (segments 8–9) at lower (e, k and q) and higher (f, l and r) magnification. Black squares specify the areas that are magnified and displayed as high magnification pictures. Scale bars: 50 µm. Immunohistochemical analysis without using IL-6 antibody was performed on epididymis from an Ncoa5+/− mouse and a representative image is shown in Supplementary Fig. S5.

Fig 3: Ultrastructure analyses of WT and Ncoa5+/− sperm. (A–D) Representative SEM analytic images of WT (A) and Ncoa5+/− epididymal sperm (B–D). Inserts are the higher magnification images of the head and neck region of the sperm. Scale bars: 5 μm (A,B); 1 μm (C,D); inserts: 1 μm. (E–H) Representative TEM analytic images of WT (E) and Ncoa5+/− epididymal sperm (F–H). Scale bars: 1μm. Cross-section of the cytoplasmic droplet (CD) of a WT spermatozoon (E). 1: Outer membrane of the CD; 2: middle piece of the sperm containing mitochondrial sheath and axoneme; 3: vacuoles within the CD; Cross-sections of Ncoa5+/− bent sperm head and neck region wrapped around by the tail (F–H). 4: nucleus; 5: middle piece containing mitochondria and axoneme wrapped around the bent head and neck; 6: disrupted mitochondria of the middle piece; 7, 8 and 9: outer membrane of the droplet; 10 and 11: acrosome; 12 and 13: wrapped-around middle piece containing mitochondria and axoneme; 14: large membranous vacuoles.

Fig 4: Representative TEM images of corpus epididymal sperm from Ncoa5+/+Il-6+/+, Ncoa5+/−Il-6+/+ and Ncoa5+/−Il-6+/− male mice. (A) TEM of Ncoa5+/+Il-6+/+ corpus epididymal sperm. Blue arrows indicate the normal morphology of the cytoplasmic droplet. (B) TEM of Ncoa5+/−Il-6+/+ corpus epididymal sperm. Red arrows indicate the abnormal morphology of sperm with the head wrapped around by the tail. (C) TEM of Ncoa5+/−Il-6+/− corpus epididymal sperm. The presence of both blue (normal) and red (abnormal) arrows indicates the number of normal sperm increases in Ncoa5+/−Il-6+/− male mice. Scale bars: 10 μm.

Fig 5: Heterozygous Il-6 deletion improves fertility and sperm morphology of Ncoa5+/− male mice. (A) Percentages of fertile Ncoa5+/+Il-6+/+ male mice or Ncoa5+/−Il-6+/− male mice bred with Ncoa5+/−Il-6+/− female mice for six months. White bar, Ncoa5+/+Il-6+/+ male mice (n = 7). Black bar, Ncoa5+/−Il-6+/− male mice (n = 6). n = the number of mice. Two-tailed Fisher’s exact test, N.S.: no significance. (B) Epididymal cauda sperm concentrations of 4-month-old Ncoa5+/−Il-6+/+ and Ncoa5+/−Il-6+/− male mice (n = 3). n = the number of mice. N.S.: no significance. (C) Percentage of motile sperm of 4-month-old Ncoa5+/−Il-6+/+ and Ncoa5+/−Il-6+/− male mice (n = 3). (D) Percentage of progressive sperm of 4-month-old Ncoa5+/−Il-6+/+ and Ncoa5+/−Il-6+/− male mice (n = 3). n = the number of mice. All values are mean ± SEM. *P < 0.05. (E,F) Representative scanning EM analysis of Ncoa5+/−Il-6+/− epididymal cauda sperm. Insert (F) is the higher magnification image of the head and neck region of the sperm. Scale bars: 5 μm; insert: 1 μm. (G,H) Representative TEM analysis of Ncoa5+/−Il-6+/− epididymal cauda sperm. Scale bars: 500 nm.