Knockout of serine-rich single-pass membrane protein 1 (Ssmem1) causes globozoospermia and sterility in male mice†

Abstract

Globozoospermia (sperm with an abnormally round head shape) and asthenozoospermia (defective sperm motility) are known causes of male infertility in human patients. Despite many studies, the molecular details of the globozoospermia etiology are still poorly understood. Serine-rich single-pass membrane protein 1 (Ssmem1) is a conserved testis-specific gene in mammals. In this study, we generated Ssmem1 knockout (KO) mice using the CRISPR/Cas9 system, demonstrated that Ssmem1 is essential for male fertility in mice, and found that SSMEM1 protein is expressed during spermatogenesis but not in mature sperm. The sterility of the Ssmem1 KO (null) mice is associated with globozoospermia and loss of sperm motility. To decipher the mechanism causing the phenotype, we analyzed testes with transmission electron microscopy and discovered that Ssmem1-disrupted spermatids have abnormal localization of Golgi at steps eight and nine of spermatid development. Immunofluorescence analysis with anti-Golgin-97 to label the trans-Golgi network, also showed delayed movement of the Golgi to the spermatid posterior region, which causes failure of sperm head shaping, disorganization of the cell organelles, and entrapped tails in the cytoplasmic droplet. In summary, SSMEM1 is crucial for intracellular Golgi movement to ensure proper spatiotemporal formation of the sperm head that is required for fertilization. These studies and the pathway in which SSMEM1 functions have implications for human male infertility and identifying potential targets for nonhormonal contraception.

Keywords: fertilization; male infertility; null mutation/knockout; sperm; spermatogenesis.

Figure 1

Ssmem1 is a conserved testis-specific gene. (A) RT-PCR in multiple tissues confirming the testis specificity of Ssmem1 in mice (Top panels) and humans (bottom panels). Hprt and GAPDH were used as controls. He, heart; Li, liver; Sp, spleen; Lu, lung; Ki, kidney; Br, brain; St, stomach; In, intestine; Te, testis; Ov, ovary; Ut, Uterus; Ep, Epididymis; H, head: B, Body; T, Tail. (B) RT-PCR from mouse testes at various postnatal days was performed. Hprt was used a control. (C) Protein sequence alignment of SSMEM1 from several mammalian species. Transmembrane region in human protein (asterisk) is well-conserved with other species. Black indicates fully conserved residues. Gray indicates conservation with residues in human.

Figure 2

Generating Ssmem1 KO mice. (A) Genomic structure of mouse Ssmem1 and scheme to generate the gene KO mice using the CRISPR/Cas9 system. Three splice variants of Ssmem1 have been reported. White and black boxes indicate untranslated and coding regions, respectively. Red arrow and black under bar indicate gRNA that targets the region. The delivery of CRISPR/Cas9 system into zygotes for mutagenesis via microinjection resulted in a 5 bp (6 bp deletion and 1 bp insertion) frameshift deletion (shown in red). (B) Genotyping of Ssmem1 alleles. Primers indicated in green color in (A) amplify specific amplicon for the WT or KO allele. (C) Mutation in amino acid sequence in KO mouse induced by—five frameshift mutation is shown in red color. Asterisk indicates a premature stop codon. The numbers above sequences are amino acid number for each. (D) Western blot analysis using testis and sperm. SSMEM1 protein is detected only in WT testis. Equal loading of total protein was confirmed by Coomassie Brilliant Blue staining.

Figure 3

Ssmem1 KO causes male infertility. (A) Average litter size from natural mating of Ssmem1 HET and KO mice. Litter size was measured by the number of pups born. Ssmem1 KO males showed complete infertile, P < 0.0001. (B) Quantification of sperm released from the cauda epididymis. There was no significant difference between HET and KO mice. (C) Images of testes from Ssmem1 HET and KO mice. (D) Average weight of individual testis. KO males showed a reduction in testis volume, P < 0.05. (E) PAS-Hematoxylin staining of testis and caudal epididymis sections from HET and KO mice. Occasional KO tubules in testis lacked germ cells (asterisks) (Scale bar, 100 μm)

Figure 4

Ssmem1 KO sperm show morphology and motility defects. (A) SEM images of HET/KO spermatozoa from caudal epididymis. KO sperm exhibited globozoospermia. (B) Sperm motility at 15 and 90 min after sperm suspension. There was a reduction in sperm motility in KO. (C) Sperm kinematic parameters measured using the CEROS II sperm analysis system. Velocities of KO sperm were lower than those of HET. P < 0.05 (^*), P < 0.01 (^**), P < 0.001 (^***).

Figure 5

Ssmem1 KO caused a delay in organelle transfer. (A) TEM images of HET/KO spermatozoa in epididymis. Nuclei showed abnormal morphology and mitochondria are located at the sperm head region, alongside the nuclei (Scale bar, 2.0 μm). (B, C) TEM images of spermatids at same stages, step 8 (B); step 9 (C) in testis. In Ssmem1 null spermatid, Golgi body (red arrows) failed to undergo migration as control spermatid at step 8. In step 9, elongating spermatids, the Golgi stayed in the caput aspect of the cell and the acrosomal membrane did not contact the cell surface (black arrowheads) in Ssmem1 null spermatids (Scale bar, 4.0 μm). (D) TEM images of sperm at step 11. Golgi and mitochondria were located at caudal region of sperm in both HET and KO sperm (Scale bar, 2.0 μm). (E) Immunostaining of HET/KO testes. The Golgi body and acrosome were stained with Golgin 97 (red) and PNA (green), respectively. Arrows indicate the Golgi retained in acrosome in mutant spermatids. Migration of Golgi from acrosome side to tail side was delayed in KO spermatids (Scale bar, 10.0 μm).

Figure 6

Schematic representation of Ssmem1 deficient spermatogenesis. The delay in organelle migration from step 8 to step 9 causes progressive changes leading to round-headed sperm in Ssmem1 KO males.