TSSK3, a novel target for male contraception, is required for spermiogenesis

Abstract

We have previously shown that members of the family of testis-specific serine/threonine kinases (TSSKs) are post-meiotically expressed in testicular germ cells and in mature sperm in mammals. The restricted post-meiotic expression of TSSKs as well as the importance of phosphorylation in signaling processes strongly suggest that TSSKs have an important role in germ cell differentiation and/or sperm function. This prediction has been supported by the reported sterile phenotype of the TSSK6 knock-out (KO) mice and of the double TSSK1/TSSK2 KO. The aim of this study was to develop KO mouse models of TSSK3 and to validate this kinase as a target for the development of a male contraceptive. We used CRISPR/Cas9 technology to generate the TSSK3 KO allele on B6D2F1 background mice. Male heterozygous pups were used to establish three independent TSSK3 KO lines. After natural mating of TSSK3 KO males, females that presented a plug (indicative of mating) were monitored for the following 24 days and no pregnancies or pups were found. Sperm numbers were drastically reduced in all three KO lines and, remarkably, round spermatids were detected in the cauda epididymis of KO mice. From the small population of sperm recovered, severe morphology defects were detected. Our results indicate an essential role of TSSK3 in spermiogenesis and support this kinase as a suitable candidate for the development of novel nonhormonal male contraceptives.

Keywords: TSSK3; evolution; fertilization; intronless gene; kinases; nonhormonal male contraceptive; sperm; spermatogenesis; testis-specific.

Figure 1.. Representative TSSK3 protein alignment based on fifteen vertebrate species.

Sequence similarities were calculated considering residue physicochemical properties (% Equivalent option; similarity threshold = 0.7). Secondary structure was inferred from Rat 2r0i.1.A Serine/threonine-protein kinase MARK2 Par-1 mutant crystal structure. A >0.7 consensus sequence is shown, where $, %, #, !, and lowercase symbols denote L/M, F/Y, N/D/Q/E/B/Z, I/V, and consensus level > 0.5, respectively. Dots in the consensus sequence represent <0.7 conservation and uppercase symbols represent total conservation, while in the alignment represent gaps.

Figure 2.. CRISPR/Cas9 generation of Tssk3 knockout mouse models.

A) Schematic representation of the Tssk3 locus in the mouse chromosome 4. The 5’ and 3’ UTR regions are depicted in gray, ORF is depicted in black. Arrowhead indicates the ORF region targeted by the sgRNA. B) Fragment of the genomic sequences of the reference WT with the sgRNA annealed, and the mutated Tssk3 KO lines. For each line, deleted bases are indicated by an open box in black. For line 2, inserted base pairs are outlined in the yellow box. Premature stop codon is indicated by a red box. Predicted protein sequences are indicated for each line (white letters over black background).

Figure 3.. Analysis of male fertility.

A) Percentage of pregnancy rates of Tssk3 WT, HET, and KO animals from Lines 1, 2, and 3 were calculated as number of pregnancies / number of plugs obtained multiplied by 100. B) Litter size indicated by number of pups per litter of Tssk3 WT, HET, and KO animals from Lines 1, 2, and 3. C) Number of sperm recovered from the cauda epididymis of Tssk3 WT, HET, and KO animals from Lines 1, 2, and 3. Data are presented as the mean + SEM. p<.02 (*), .01 (**), or .001 (***). D) Quantification of sperm morphological defects of Tssk3 WT, HET, and KO animals from Lines 1, 2, and 3. Sperm with malformed heads or broken tails were counted as defective. N = number of independent replicates, n = total number of sperm counted.

Figure 4.. Sperm morphology.

A) Representative phase contrast images combined with nuclear (Hoechst in blue) and acrosome (PNA in green) staining indicating the morphology of sperm from Tssk3 WT, HET, and KO animals from Lines 1, 2, and 3. Scale bar = 20 μm. Insets of the KO images are displayed in the right panel. Scale bar = 5 μm. B) Transmission electron microscopy analyses of sperm cells from WT and KO mice (line 1). (i) Cross sections of sperm flagella show normal distribution of axoneme and fibrous sheath components in WT mice. (ii) Black arrows indicate loss of axoneme and outer dense fiber components in Tssk3 KO sperm. (iii) Detached acrosome was observed in KO sperm indicated by the arrow. (iiii) Abundant round cells were detected in sperm samples from KO mice.

Figure 5.. TSSK3 deletion in testis alters late spermiogenesis.

A) Representative anatomical images of testis collected from WT and TSSK3 KO mice (lines 1, 2 and 3). Scale bar = 250 μm. B) Quantification of testis weight collected from WT and Tssk3 KO animals, N= 11. Statistical comparison of WT and KO testis weights using a one-way ANOVA, followed by Tukey’s test, **p <0.01. C) Representative images of PAS-hematoxylin-stained testis-sections of WT and KO stage VII/VIII testis sections. Adjacent inset image represents a magnified view of the corresponding region marked with the rectangular box. Arrowhead: step 7/8 spermatids; asterisk: step 15/16 elongated spermatids; arrow: pre-leptotene and leptotene cells. Scale bar: 20 μm, n= 3.

Figure 6.. Localization of TSSK3 in mouse sperm.

A) Western blot of sperm proteins from Tssk3 WT, HET and KO animals from the three lines. A specific band detected by an anti-TSSK3 antibody is indicated by an arrow at ~30 KDa. B) Immunofluorescence of WT and KO (Line 1) sperm. Representative images of difference interference contrast (DIC, left panel), and epifluorescence indicating TSSK3 localization in green (right panel). Scale bar 20 μm. C) 3D-SIM image indicating the localization of TSSK3 (green) and acrosome staining with PNA (red) in WT sperm (left panel). Arrow indicates site of the cross-section displayed in right panel. Scale bar 1 μm. Cross-section of the sperm tail indicating the central localization of TSSK3 (right panel). Cross-section scale bar 0.25 μm.