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. 2019 Feb 12;10(2):133. doi: 10.3390/genes10020133

Figure 6.

Figure 6

Flag-Sly1 SP4 transgenic line generated and analyzed independently confirms that the rescue of Sly1 expression does not lead to the rescue of the spermiogenic phenotype of mice with NPYq deficiency. (A) Gene expression in testes from NPYq-deficient mice without (2/3NPYq-) and with (2/3NPYq- tsgic) the Flag-Sly1 SP4 transgene was obtained by real-time RT-PCR with Acrv1 as a loading control and normalized to wild-type XY controls. The graphs are mean ± SEM with n = 3. Statistical significance (t-test): * < 0.05; *** < 0.001, in comparison with NPYq-deficient mice with XY; the difference between transgenics and their negative siblings is marked with a horizontal lane above the relevant graph pairs. Primer sequences are shown in Table S2. (B,C) SLY1 expression rescue shown with Western blot performed with whole testes lysates obtained from males with moderate NPYq deficiency (2/3NPYq-) (B) or from males lacking the entire Y chromosome long arm (NPYq-) (C) with (tsgic SP4) and without (neg sib) the addition of the Flag-Sly1 SP4 transgene. Levels of protein expression were quantified with ImageJ software and normalized to Ponceau (PON) signal. The data represent an average ± SDev, with n = 2–3. Statistical significance (t-test, p < 0.05): bars marked with different letters are significantly different. D: Immunofluorescence detection of transgenic SLY1 protein (green) on stage VII testicular tubules from mice lacking the entire Y chromosome long arm without (NPYq-) and with (NPYq- tsgic) the Flag-Sly1 SP4 transgene. Lectin (red) was used to stage the tubules and DAPI (blue) was used to stain the nuclei. Bar = 10 µm. (E) Sperm headshape analysis performed for NPYq-deficient mice without (2/3NPYq-) and with (2/3NPYq- tsgic) the Flag-Sly1 SP4 transgene, and XY controls. Normal headshape and three categories of headshape defects were differentiated and quantified. The data represent an average ± SEM with n = 13, 8, and 3 males of the XY, 2/3NPYq-, and 2/3NPYq- tsgic genotype; a total of 1227, 651, and 274 sperm were examined for XY, 2/3NPYq-, and 2/3NPYq- tsgic genotype, respectively. Statistical significance: two-way ANOVA with genotype and sperm headshape as factors revealed a significant effect of genotype (p = 0.0034), headshape (p < 0.0001) and interaction (p < 0.0001). The results of paired comparison for specific sperm headshape between transgenic and negative siblings in post-hoc Bonferroni test are shown within graphs: a p < 0.05 when compared to XY and b p < 0.05 when compared to 2/3NPYq-.