Homozygous Loss of Septin12, but not its Haploinsufficiency, Leads to Male Infertility and Fertilization Failure

Abstract

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.

Keywords: SEPTIN12; calcium oscillation; fertilization failure; male infertility; oocyte activation.

FIGURE 1

Male infertility of Septin12 ^−/− mice. (A) Sequencing chromatograph showing the c.72C>A mutation on SEPTIN12 in the male patient. (B) Schematic illustration of the strategy for knocking out the Septin12 gene by CRISPR/Cas9. Genotyping primers F1, R1, and R2 are marked by arrows. (C) Genotyping of WT, Septin12 ^+/−, and Septin12 ^−/− mice by PCR. A 371 bp band is amplified from the WT allele with primers F1 and R1, while a 608 bp band is amplified from the KO allele with primers F1 and R2. (D) Male fertility of WT, Septin12 ^+/−, and Septin12 ^−/− mice.

FIGURE 2

Abnormal sperm morphology and motility in Septin12 ^−/− male mice. (A) The morphology of the testis from WT, Septin12 ^+/−, and Septin12 ^−/− mice. (B) Testis weight of WT, Septin12 ^+/−, and Septin12 ^−/− mice (n = 5 for each genotype). NS stands for not statistically significant. (C,D) Hematoxylin and eosin staining of testis (C) and epididymis (D) sections from WT, Septin12 ^+/−, and Septin12 ^−/− mice. Scale bars, 100 μm. (E–H) Sperm count (E), morphology (F,G), and motility (H) of WT, Septin12 ^+/−, and Septin12 ^−/− mice (n = 5 for each genotype). Sperms were isolated from the epididymis. The data are represented as means ± SD, ***p < 0.001. (F) Scale bars, 10 μm. (I) Ca²⁺ oscillation profiles of ICSI embryos with sperms from WT mice (left panel) or Sept12 ^−/− mice (right panel). The numbers in parentheses denote the fraction of embryos presented in the plots. The numerator is the number of ICSI embryos represented in the plot from two independent experiments, and the denominator is the total number of ICSI embryos from two independent experiments.

FIGURE 3

Disturbed distribution of PLCζ and acrosome malformation in Septin12 ^−/− spermatozoa. (A,B) Immunofluorescence staining of PLCζ (A) and SEPTIN12 (B) in spermatozoa isolated from the epididymis of WT, Septin12 ^+/−, and Septin12 ^−/− mice. Scale bars: 10 μm. (A) The PLCζ signal at the acrosome region is marked by solid white triangle, and the dashed triangle shows the lack of PLCζ signal at the acrosome region of Septin12 ^−/− spermatozoa. (B) The signals of SEPTIN12 at the neck and annulus are indicated by solid white arrows and triangles, respectively. Dashed arrow and triangle point to the neck and annulus without SEPTIN12 signal. (C,D) Immunofluorescence staining of PLCζ (C) and acrosome (D) in testis sections of WT, Septin12 ^+/−, and Septin12 ^−/− mice. Enlarge images are shown in the right side. Scale bars: 50 μm. (D) The acrosome was stained by Alexa Fluor 488-lectin-PNA. (E) Immunofluorescence staining of acrosome in spermatozoa isolated from the epididymis of WT, Septin12 ^+/−, and Septin12 ^−/− mice. Scale bar: 10 μm.