RABL-regulated pathways: a new tale in sperm function.

M ammalian sperm have a specialized motile flagellum to provide the unidirectional propulsion required to achieve fertilisation of the egg. Defective flagella development and/or function is a significant cause of reduced sperm motility (asthenospermia) and male infertility. Over the past decade, a growing number of distinct proteins with crucial roles in sperm flagella have been identified by genetic loss-offunction approaches in mice, in which the deliberately engineered or spontaneous mutation of individual genes have lead to anomalies in sperm tail assembly, structure and function. In a recent study published in PLoS Genetics, Lo et al. have utilized random mutagenesis induced by the chemical mutagen N-ethyl-nitrosourea to identify a previously uncharacterized protein (RABL2) in murine sperm flagella required for male fertility. By combining the N-ethyl-nitrosoureainduced random mutagenesis with phenotypic screening of fertility, Lo et al. identified a mouse line displaying recessive male infertility associated with a nucleotide mutation localized to the autosomal Rabl2 gene. The genomic Rabl2 mutation was an A to G nucleotide substitution predicted to cause a single amino-acid (D73G) substitution in two putative RABL2 protein isoforms derived from alternatively spliced Rabl2 transcripts. Structural differences between these predicted RABL2 isoforms (designated 1 and 2) were not described, but each would contain distinct amino acid sequences in their C-terminal region. Sterile males homozygous for the Rabl2 mutation (Rabl2) exhibited mature testes 85% of normal size which produced 50% less sperm. In addition to oligospermia, epididymal sperm length was 17% shorter than normal, and functional analysis revealed an 83% reduction in progressive sperm motility. Female mice carrying the Rabl2 mutation remained fertile. The oligoasthenospermia observed in infertile Rabl2 males suggested that the Rabl2 gene encodes a factor essential for the functional development and motility of mouse spermatozoa. Lo et al. found that Rabl2 isoform 1 and 2 transcripts were enriched in male germ cells, and detected in many other tissues, including the ovary and those containing motile flagella/cilia (interchangeable terms), such as brain, kidney, lung and trachea. Further characterisation of RABL2 was directed to isoform 2, possibly due to its higher mRNA expression levels during testicular development compared to isoform 1. Two human homologues, RABL2B and a paralogue RABL2A encoding a predicted protein with four amino acid changes, have been previously identified. Human RABL2B shares 89% identity with murine RABL2 isoform 2. While the function of human RABL2 homologues are not known, gene expression databases (EST) suggested RABL2A/RABL2B are expressed in a wide range of tissues including testis, brain, kidney and uterus. Inspection of another gene expression database (NCBI GEO Profiles) for human RABL2A/RABL2B supports a broad tissue expression pattern, including the testis, and further suggests both genes are highly expressed in epididymis and trachea (http://www.ncbi.nlm.nih.gov/ geoprofiles: RABL2A: GDS596, GDS829, GDS3113; RABL2B: GDS832), consistent with roles in spermatozoa and cilia containing tissues. Mouse Rabl2 encodes an evolutionarily conserved member of the Rab-like Ras GTPase superfamily. Previous studies have shown that related Rab-like proteins have key roles in flagella development and function in diverse species including Chlamydomonas reinhardtii (green algae) and Trypanosoma brucei (flagellate protozoan). The murine Rabl2 asthenospermia phenotype suggested that conserved RABLmediated actions extend to mammalian motile flagella. The assembly and function of motile flagella requires bidirectional intraflagella transport (IFT) of large protein complexes along a central microtubule-based structure known as the axoneme. Delivery of cargo proteins along the flagella axoneme involves components of IFT complex A or complex B machinery. In Chlamydomonas, targeted RNA interference showed that the Rab-like protein RABL4 (IFT27) forms part of the IFT complex B during flagella tip development. Knock-down studies of RABL5 (IFT22) in Trypanosoma brucei produced stunted flagella packed with IFT proteins. Predicting that mammalian RABL2 may exhibit related functions, Lo et al. used immunoprecipitation to show that RABL2 interacts with complex B components including RABL4 (IFT27), IFT81 (Cdv1) and IFT172. Immunofluorescent microscopy colocalized RABL2 with these IFT proteins in the mid-piece of elongated spermatids in the testis. It was not reported if the mutated RABL (D73G) had diminished interaction with these IFT proteins, but the wild-type RABL2-IFT protein interactions combined with the Rabl2 phenotype indicated that RABL2 has a specific role in IFT particles. RABL2 contains the five consensus motifs characteristic of GTP/GDP binding in the Ras superfamily, suggesting that it may cycle between GTP-active and GDP-inactive states. The Rabl2 D73G mutation occurred in a b-sheet domain, which in other superfamily members is required for interactions with other proteins, including factors coordinating GDP–GTP exchange. To study GTP/GDP involvement in RABL2 function, Lo et al. used recombinant RABL2 isoform 2 to identify proteins in testis extracts that preferentially bound to RABL2 in a GTP-bound versus GDP-bound state. Mass spectrometry identified 89 proteins that interacted with the putative active state (GTP-bound) RABL2. ANZAC Research Institute, Concord Hospital, University of Sydney, Sydney, NSW 2139, Australia Correspondence: Dr CM Allan ([email protected]) Asian Journal of Andrology (2013) 15, 87–88 2013 AJA, SIMM & SJTU. All rights reserved 1008-682X/13 $32.00