Testis-specific serine kinase 3 is required for sperm morphogenesis and male fertility

Abstract

Background: The importance of phosphorylation in sperm during spermatogenesis has not been pursued extensively. Testis-specific serine kinase 3 (Tssk3) is a conserved gene, but TSSK3 kinase functions and phosphorylation substrates of TSSK3 are not known.

Objective: The goals of our studies were to understand the mechanism of action of TSSK3.

Materials and methods: We analyzed the localization of TSSK3 in sperm, used CRISPR/Cas9 to generate Tssk3 knockout (KO) mice in which nearly all of the Tssk3 open reading frame was deleted (ensuring it is a null mutation), analyzed the fertility of Tssk3 KO mice by breeding mice for 4 months, and conducted phosphoproteomics analysis of male testicular germ cells.

Results: TSSK3 is expressed in elongating sperm and localizes to the sperm tail. To define the essential roles of TSSK3 in vivo, heterozygous (HET) or homozygous KO male mice were mated with wild-type females, and fertility was assessed over 4 months; Tssk3 KO males are sterile, whereas HET males produced normal litter sizes. The absence of TSSK3 results in disorganization of all stages of testicular seminiferous epithelium and significantly increased vacuolization of germ cells, leading to dramatically reduced sperm counts and abnormal sperm morphology; despite these histologic changes, Tssk3 null mice have normal testis size. To elucidate the mechanisms causing the KO phenotype, we conducted phosphoproteomics using purified germ cells from Tssk3 HET and KO testes. We found that proteins implicated in male infertility, such as GAPDHS, ACTL7A, ACTL9, and REEP6, showed significantly reduced phosphorylation in KO testes compared to HET testes, despite unaltered total protein levels.

Conclusions: We demonstrated that TSSK3 is essential for male fertility and crucial for phosphorylation of multiple infertility-related proteins. These studies and the pathways in which TSSK3 functions have implications for human male infertility and nonhormonal contraception.

Keywords: knockout mouse; oligoteratozoospermia; oligozoospermia; proteomics; teratozoospermia.

Figure 1.. Generating Tssk3 KO mice.

A. Multi-tissue RT-PCR expression analysis of TSSK3 in humans. GAPDH was used as a loading control. Li, Liver; Ki, Kidney; Br, Brain; In, Intestine; Te, Testis; Ut, Uterus; Ep, Epididymis; H, Head (Caput); B, Body (Corpus); T, Tail (Cauda). B. Multi-tissue RT-PCR expression analysis of Tssk3 in mice. Hprt was used as a loading control. He, Heart; Li, Liver; Sp, Spleen; Lu, Lung; Ki, Kidney; Br, Brain; St, Stomach; In, Intestine; Te, Testis; Ov, Ovary; Ut, Uterus; Ep, Epididymis. C. RT-PCR from mouse testes at various postnatal days. D. Heatmap depicting RNA-seq-based Transcripts Per Million (TPM) values for mouse Tssk3 in the indicated reproductive tissues and cells. Dync1i2 was used a “loading” control. White = 0, Black = Max. E. Genomic structure and strategy of generating Tssk3 KO mice, and the genetic sequences of mouse Tssk3 deleted by the CRISPR/Cas9 system. F. Genotyping of Tssk3 alleles. Primers shown in Fig. 1E amplify specific amplicons for the WT or KO alleles. The sizes of several DNA ladder bands are shown for comparison. G. Western blot analysis using testis, sperm, and spermatozoa fractionated into sperm heads and sperm tails. TSSK3 protein is detected in the testes from WT and HET, and whole sperm and sperm tails from WT mice.

Figure 2.. Tssk3 KO causes male sterility.

A. Average litter size from natural mating of Tssk3 HET and KO mice. Litter size was measured by the number of pups born. Tssk3 KO males showed complete infertility. P<0.00001. B. The average weight of individual testes and images of testes from Tssk3 HET and KO mice. ns, not significant. C. Quantification of sperm released from the cauda epididymis. Tssk3 KO males showed a significant reduction in testis volume, P<0.0001. D. Images of Tssk3 HET and KO spermatozoa from caudal epididymis. KO sperm exhibited abnormal sperm morphology. Scale bar, 20 μm.

Figure 3.. Histological and ultrastructural analysis of testis sections from control and Tssk3 KO mice.

PAS-Hematoxylin staining of testis from Tssk3 HET (A) and KO (B) mice. Representative tubule cross-sections from each stage (I-XII) as indicated in the white box in the upper left-hand corner of each image. Higher magnification insets for Stage IV-VI are included as indicated with blue boxes to better show vacuolization present in KO (B), but not HET (A) mice. Abnormalities observed in the Tssk3 KO (B) mice are indicated as follows: a, elongated spermatids not fully condensed; b, disorganized appearance of elongated spermatids; c, significant vacuolization present; d, elongated spermatids embedded in epithelium (not lining the lumen), vacuolization; e, unreleased elongated spermatids, vacuolization; f, a mixture of early elongating spermatids and unreleased fully elongated spermatids, vacuolization; g, a mixture of early elongating spermatids and unreleased fully elongated spermatids; h, a mixture of developing (not fully condensed) elongated spermatids and unreleased fully elongated spermatids; i, a mixture of developing (not fully condensed) elongated spermatids and unreleased fully elongated spermatids. Scale bar, 50 μm.

Figure 4.. Histological of epididymis sections and ultrastructural analysis of testis sections from control and Tssk3 KO mice.

A. PAS-Hematoxylin staining of epididymis from Tssk3 HET and KO mice. Scale bars as indicated. B. Transmission electron microscopy images of Tssk3 HET and KO testis. (a) HET and (d) KO spermatids at step 9 (Scale bar, 2 μm). Spermatozoa at step 14–15 from (b) HET and (e) KO (Scale bar, 2 μm). Tssk3 KO sperm have cytoplasm around normal-shaped nuclei (n). The magnified images, (c) and (f) show perspectives in white squares on (b) and (f), respectively. KO sperm have detached acrosome (ac, red arrows) from the sperm nuclei (n) (Scale bar, 2 μm).

Figure 5.. Phosphoproteomic analysis to uncover putative TSSK3 substrates.

A. Volcano plot of quantitative analysis of proteins identifies MS profiling. Proteins expressed with fold-changes (HET: KO ratio) > 1.25. (p < 0.05) were selected. B. Volcano plot of quantitative analysis of phosphopeptides identified by MS. Phospho-proteins with fold-changes (HET: KO ratio) > 1.25. (p < 0.05) were selected. C. Logo motif of the phosphosites. The top 100 phosphosites phosphorylated at higher levels in Tssk3 HET than in KO testes were subjected to the sequence logos creation using PhosphoSitePlus^®. D. The lists of upregulated or phosphorylated proteins in HET compared to KO, that are reported in each mutant mouse model show infertility/subfertility phenotype.