RAB-like 2 has an essential role in male fertility, sperm intra-flagellar transport, and tail assembly

Abstract

A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2) as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT) proteins and delivers a specific set of effector (cargo) proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.

Figure 1. The Mot mouse line contains a point mutation in the Rabl2 gene.

(A) The Mot line contains a single A to G substitution in the first codon of exon 5 which would affect RABL2 isoforms (transcript) 1 and 2 but not the putative isoform 3. Boxes indicate exons (white are non-coding sequences and black are protein coding sequences), lines indicate introns. The arrow indicates the position of the promoter and the transcriptional direction. ATG indicates the translation initiation site. (B) The Rabl2^Mot/Mot mutation results in the conversion of an evolutionarily conserved aspartic acid (D) into a glycine (G) (arrow). (C) RABL2 contains all five consensus motifs involved in GTP binding in RAB proteins. The consensus motif sequence is indicated below each shaded box and the actual RABL2 sequence indicated in the box. The asterisk indicates the position of the Mot mutation.

Figure 2. Rabl2 expression and localization.

(A) The adult mouse testis expresses mRNA for Rabl2 isoform 1 and isoform 2, of which isoform 2 is more highly expressed. N = 3 mice per genotype (*** p<0.001). Relative Rabl2 isoform 1 (B) and isoform 2 (C) expression in the developing post-natal testis indicates that both isoforms are predominantly produced within haploid germ cells which first appear at day 20 post-natal. The day zero value was set to 1 and all other ages expressed relative to this value. (D) RABL2 localization (red) within wild type and Mot homozygous mutant testis tissue and caudal epididymal sperm. The inset indicates staining obtained when the primary antibody was pre-absorped with excess immunizing peptide prior to immunofluorescence. The white arrows indicate the position of the mid-piece of the sperm tail. The yellow arrow indicates the position of the principal piece. DNA was labelled using DAPI (blue). The relative expression of Rabl2 isoform 1 (E) and 2 (F) in adult tissues. N = 3 mice per group. The testis was set at 1 and all other tissues expressed relative to this value.

Figure 3. The sterility phenotype observed in Rabl2^Mot/Mot males.

Testis histology from a 10 weeks old wild type (A) and Rabl2^Mot/Mot (B) male. Sections were stained with PAS. Spermatogenesis appeared qualitatively normal. Scale bars represent 100 µm. (C) Testis weights were significantly reduced in Rabl2^Mot/Mot (Mut) animals compared to wild type (WT) littermates. (D) Daily sperm output from 10 weeks old wild type (WT) and Rabl2^Mot/Mot (Mut) males. Haematoxylin and eosin stained sperm from wild type (WT) (E) and Rabl2^Mot/Mot (Mut) (F) animals revealed no obvious differences between genotypes. Scale bars represent 25 µm (G) Sperm from 9 weeks old Rabl2^Mot/Mot (black) however, had a significantly compromised ability for any form of motility and a very pronounced defect in the capacity for progressive motility compared to sperm from wild type (white) littermates. (H) Sperm from four different wild type and three different Rabl2^Mot/Mot males (one mouse per lane) probed for RABL2 protein content. Protein loading was normalized using the sperm head protein ACRBP. (J–K) Electron microscopy of a cross-section of the mid-piece of a sperm from a wild type (WT) (I) and Rabl2^Mot/Mot male (J) revealed no discernable difference in ultrastructure. (I) The length of sperm tails from wild type (WT) and Rabl2^Mot/Mot (Mut) males. (* p<0.05, ** p<0.01, *** p<0.001).

Figure 4. RABL2 binds to components of IFT complex B.

(A) Immunoprecipitation of IFT complex B components (IFT27, IFT81 and IFT172) from testis extracts showed that all three were bound to RABL2. Ig = an immunoglobulin isotype and concentration matched control. −/+ indicate the presence of exogenous GTP in the testis homogenate to explore the potential regulation of binding to RABL2 by GTP binding status. (B–E) The co-localization of RABL2 (red) in the mid-piece of elongating spermatids (white arrows) within the seminiferous epithelium with each of IFT27 (B), IFT81 (C) and IFT172 (D). (E) A representative primary antibody control wherein the primary antibody was omitted from the staining protocol. Scale bars equal 25 µm.

Figure 5. The identification of RABL2 effector proteins.

(A) Preferential binding of candidate effector proteins to GTP-RABL2 (active state) over GDP (inactive state) was confirmed by Western blotting of additional affinity column eluates. (B) Specific binding of effector proteins to RABL2 was confirmed by immunoprecipitation of effector proteins (EP) from testis homogenates then probing for binding to RABL2 in Western blots. The specificity of immunoprecipitations was confirmed using parallel reactions wherein the precipitating antibody was replaced by isotype and concentration matched immunoglobulin.

Figure 6. The Mot mutation resulted in the decreased delivery of effector proteins into sperm tails.

The immunolocalization of RABL2 effector proteins (red) in sperm from wild type and Rabl2^Mot/Mot mice. DNA was labelled with DAPI. The percentage change in total sperm content of individual effector proteins was quantitated using Western blotting blotting (right hand panel, upper bands). Sperm protein loading was normalized to sperm head protein ACRBP (right hand panel, lower bands). Each Western blot was done a total of three times and results averaged. This figure contains representative images. The approximate percentage change (rounded to the nearest 10% to reflect the non-linearity of ECL detection methods) and the p values are indicated. N = 3 animals per genotype per effector protein.