SPATS1 (spermatogenesis-associated, serine-rich 1) is not essential for spermatogenesis and fertility in mouse

Abstract

SPATS1 (spermatogenesis-associated, serine-rich 1) is an evolutionarily conserved, testis-specific protein that is differentially expressed during rat male meiotic prophase. Some reports have suggested a link between SPATS1 underexpression/mutation and human pathologies such as male infertility and testicular cancer. Given the absence of functional studies, we generated a Spats1 loss-of-function mouse model using CRISPR/Cas9 technology. The phenotypic analysis showed no overt phenotype in Spats1-/- mice, with both males and females being fertile. Flow cytometry and histological analyses did not show differences in the testicular content and histology between WT and knockout mice. Moreover, no significant differences in sperm concentration, motility, and morphology, were observed between WT and KO mice. These results were obtained both for young adults and for aged animals. Besides, although an involvement of SPATS1 in the Wnt signaling pathway has been suggested, we did not detect changes in the expression levels of typical Wnt pathway-target genes in mutant individuals. Thus, albeit Spats1 alteration might be a risk factor for male testicular health, we hereby show that this gene is not individually essential for male fertility and spermatogenesis in mouse.

Fig 1. Conservation of SPATS1 in metazoans.

(A) Highly resolved tree showing the presence of SPATS1 and its phylogenetic relationship across different metazoans taxa. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. (B) Alignment of SPATS1 amino acidic sequence from mouse (Mus musculus) and man (Homo sapiens). Alignment was performed with ClustalW and BoxShade. Identical amino acids are highlighted in black and similar amino acids in grey.

Fig 2. Genomic structure and knockout strategy of Spats1.

(A) Graphical representation of the structure of mouse Spats1. Coding exons appear as solid black bars and non-coding exons as white bars. Introns are represented as horizontal lines. Sequence of the sgRNA, designed to target exon 3, is underlined in black, and the PAM sequence is underlined in red. (B) Representative genotyping results obtained through sequencing of PCR products amplified from mouse tail tips. The 2 bp deletion in Spats1^-/- mutants is shown. (C) Western blot analysis showing the detection of SPATS1 in testicular lysates of two WT mice, but not in those of 2 KO ones. β-actin was used as a loading control.

Fig 3. Fertility and spermatogenesis analyses of Spats1^-/- young adult mice of 45–60 dpp.

(A) Number of offspring per litter, obtained by intercrossing WT individuals, crossing WT females with KO males, WT males with KO females, and KO males and females. (B) Representative images of testes from WT and Spats1^–/–male mice (i), and comparison of testicular diameter (ii) and weight (iii) of WT, Spats1^+/–, and Spats1^–/–animals. (C) Flow cytometric (FCM) analysis of testicular cell suspensions from WT, Spats1^+/–, and Spats1^–/–mice. Representative FCM profiles (histograms and dot plots) from WT and Spats1^–/–are shown below. (D) Histological analyses of the testes of WT and Spats1^–/–mice. Scale bar in the upper images corresponds to 50 μm, while scale bar in the lower images corresponds to 25 μm. (E) Total motility (percentage) of sperm from cauda epididymis of WT and KO mice. Non-capacitated and capacitated sperm were analyzed by CASA. (F) Percentage of sperm from cauda epididymis of WT and KO mice with progressive motility, as assessed by CASA. Again, non-capacitated and capacitated sperm are shown. (G) Percentage of sperm from cauda epididymis with normal head morphology, from WT and KO mice. All the data correspond to the analysis of 3–6 animals of each type, and are presented as the means ± SD.

Fig 4. Fertility and spermatogenesis analyses of Spats1^-/- 1 year old mice.

(A) Average litter size, obtained by intercrossing WT individuals, or crossing KO males with WT females. (B) Histological analyses of the testes of WT and Spats1^–/–male mice. Scale bar: 25 μm. (C) Total motility (percentage) of sperm from cauda epididymis of WT and Spats1^–/–mice, as assessed by CASA. Non-capacitated and capacitated sperm were analyzed. (D) Percentage of sperm with progressive motility, from the cauda epididymis of WT and KO mice. (E) Percentage of sperm from the cauda epididymis with normal head morphology, from WT and KO mice. All the data correspond to the results obtained from the analysis of 3–6 individuals of each type, and are presented as the means ± SD.

Fig 5. Comparative expression analysis of genes of the Wnt pathway in Spats1^-/- and WT mice.

(A) qRT-PCR of testicular RNA from 55 dpp WT and KO mice. (B) qRT-PCR of testicular RNA from 1 year-old WT and KO mice. Data are expressed as absolute normalized expression levels in arbitrary units (mean ±SD). Lef1: Lymphoid enhancer binding factor 1; Tcf1: Transcription factor 1; Ccnd1: Cyclin D1; c-Myc: Myc proto-oncogene; Dvl2: Dishevelled-2; Dvl1: Dishevelled-1; Ctnnb1: β-catenin; Wnt4: Wingless family member 4.