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The SEPTIN12 gene has been associated with male infertility. Male Septin12+/− chimera mice were infertile, supporting the prevailing view that SEPTIN12 haploinsufficiency causes male infertility. In this study, we identified a heterozygous mutation on SEPTIN12, c.72C>A (p.Cys24Ter) in the male partner of a patient couple, who had a previous fertilization failure (FF) after intracytoplasmic sperm injection (ICSI) and became pregnant after ICSI together with artificial oocyte activation (AOA). To investigate the role of SEPTIN12 in FF and oocyte activation, we constructed Septin12 knockout mice. Surprisingly, Septin12−/− male mice, but not Septin12+/− male mice, are infertile, and have reduced sperm counts and abnormal sperm morphology. Importantly, AOA treatment enhances the 2-cell embryo rate of ICSI embryos injected with Septin12−/− sperm, indicating that FF caused by male Septin12 deficiency is overcome by AOA. Mechanistically, loss of PLCζ around the acrosome might be the reason for FF of Septin12−/− sperm. Taken together, our data indicated that homozygous knockout of Septin12, but not Septin12 haploinsufficiency, leads to male infertility and FF.

Additional file 2. Additional results figures. Figures S1-S12 show additional data related to the results shown in the main figures. Figure S1. The plasmid construction process and plasmid maps. A, pcDNA3.1( +)-CYP1B1 plasmid construction diagram. B-D, Plasmid maps of pcDNA3.1( +)-CYP1B1, CRISPRi-CYP1B1 and CRISPRi-Lac. Figure S2. Transfection efficiency/cytotoxicity of the plasmids. pcDNA3.1( +) plasmids, which expresses 3 �� flag-tagged peptide, were transfected into LNCaP cells. The transfection efficiency (A) and cytotoxicity (B) of the plasmids were assessed. Scale Bar = 50 ��m. Figure S3. Selected ion monitoring (SIM) chromatograms of the 4-OHE2 standard. E2 was dissolved in methanol. The monitored ion in the mass spectrum had a mass/charge ratio of 755 m/z and a retention time of 6.95 min. Figure S4. Validation of CYP1B1 silencing efficiency in LNCaP-abl and PC3 cells and CYP1B1 overexpression efficiency in LNCaP cells by western blotting. Figure S5. HPLC���MS assay results showing the concentration of intracellular 4-OHE2 in CYP1B1 knockdown or control PC3 cells (t-test). *p

Additional file 3. Supplementary Figures. Fig. S1. (A) Representative IF graphs of NRF2 in LNCaP abl cells treated with DMSO or 10 nM E2 treatment; scale bar, 50 μm. (B) Western blot analysis showing LNCaP abl or PC3 with NRF2 knockdown. (C) qPCR analysis showing expression changes of the indicated genes in LNCaP abl and PC3 treated with either DMSO or 10 nM E2 (one-way ANOVA). All studies were repeated at least three times. The data are presented as the mean ± SEM. * P < 0.05, **P < 0.01. Fig. S2. (A) qPCR analysis of the indicated in LNCaP abl or PC3 cells with or without ICI182780 after treatment with either DMSO or 10 nM E2 (one-way ANOVA). (B) qPCR and Western blot analysis of LNCaP, LNCaP abl, and PC3 cells transfected with siAR and siCtrl. qPCR analysis showing expression changes of NRF2(t-test). (C) qPCR and Western blot analysis were conducted in LNCaP abl and PC3 cells transfected with siERα, siERβ, siGPR30 and siCtrl. qPCR analysis showing expression changes of NRF2(t-test). All studies were repeated at least three times; the values are the mean ± SEM; *P < 0.05, **P < 0.01. Fig. S3. Scratch test was conducted in PC3 cells with different treatments, scale bar, 200 μm. All studies were repeated at least three times; the values are the mean ± SEM; *P < 0.05. Fig. S4. The mRNA-seq analysis indicated that the expression of antiapoptotic genes was up-regulated in bicalutamide resistant cells and positively correlated with the expression of NRF2. The heatmap was generated by an online analytic tool ( www.xiantao.love ).

Additional file 1. Supplementary description of cell lines. In this additional file, the characteristics and culture methods of bicalutamide sensitive cells and resistant cells, as well androgen-dependent and independent cells were detailly described.

Additional file 1. Additional protocols tables. Tables S1-S3 show additional data related to the methods of the study. Table S1. Primers used for qRT-PCR. Table S2. Primary antibodies used in the western blotting and IF assays. Table S3. Primers used for plasmid construction.

Additional file 2. Supplementary Tables. Table S1: shNRF2 sequences. Table S2: Primer sequences. Table S3: List of antibodies. Table S4: Plasmids sequences.