The Caenorhabditis elegans homologue of deleted in azoospermia is involved in the sperm/oocyte switch

Abstract

The Deleted in Azoospermia (DAZ) gene family encodes putative translational activators that are required for meiosis and other aspects of gametogenesis in animals. The single Caenorhabditis elegans homologue of DAZ, daz-1, is an essential factor for female meiosis. Here, we show that daz-1 is important for the switch from spermatogenesis to oogenesis (the sperm/oocyte switch), which is an essential step for the hermaphrodite germline to produce oocytes. RNA interference of the daz-1 orthologue in a related nematode, Caenorhabditis briggsae, resulted in a complete loss of the sperm/oocyte switch. The C. elegans hermaphrodite deficient in daz-1 also revealed a failure in the sperm/oocyte switch if the genetic background was conditional masculinization of germline. DAZ-1 could bind specifically to mRNAs encoding the FBF proteins, which are translational regulators for the sperm/oocyte switch and germ stem cell proliferation. Expression of the FBF proteins seemed to be lowered in the daz-1 mutant at the stage for the sperm/oocyte switch. Conversely, a mutation in gld-3, a gene that functionally counteracts FBF, could partially restore oogenesis in the daz-1 mutant. Together, we propose that daz-1 plays a role upstream of the pathway for germ cell sex determination.

Figure 1.

DAZ homologues from three Caenorhabditis species. (A) Alignment of the deduced amino acid sequences of Cb-DAZ-1, Cr-DAZ-1, and C. elegans DAZ-1. Black boxes indicate identical residues and shaded boxes indicate similar residues. The RRM is underlined and amino acid residues conserved in the BOULE subfamily proteins are marked with asterisks. The putative DAZ motif is double underlined. (B) Exon–intron structures of Cb-daz-1, Cr-daz-1, and daz-1. Exons are shown by boxes, introns, by broken lines, and 5′ and 3′ UTR sequences, by lines. The RRM motif is indicated by split black boxes, and the putative DAZ motif, by a shaded box, in each gene.

Figure 2.

RNAi for Cb-daz-1 and Cr-daz-1. (A, B, E, and F) Gonads were dissected from the following adult animals and stained with DAPI. (C and D) Whole animals were stained with DAPI. (A) C. briggsae wild-type hermaphrodite with no treatment. (B) C. briggsae Cb-daz-1(RNAi) F1 hermaphrodite. (B) C. briggsae P0 hermaphrodite with mock RNAi treatment. (D) C. briggsae Cb-daz-1(RNAi) P0 hermaphrodite. (E) C. remanei wild-type female with no treatment. (F) C. remanei Cr-daz-1(RNAi) F1 female. Arrows indicate germ nuclei at the diakinesis stage during oogenesis. Arrowheads indicate the distal end of each gonad. Bar, 50 μm.

Figure 3.

Synthetic phenotype of the C. elegans daz-1 mutation or RNAi and the fem-3(q20gf) mutation. (A–E) DAPI-stained images of extruded gonads from adult hermaphrodites are shown. (A) Wild-type C. elegans at 20°C. (B) The daz-1 mutant at 20°C. (C) The fem-3(q20gf) mutant at 15°C. (D) The fem-3(q20gf) mutant at 25°C. (E) The daz-1; fem-3(q20gf) double mutant at 15°C. (F and G) Whole mount DAPI-stained images of adult hermaphrodites cultured for 5 d at 15°C after RNAi treatment. (F) Wild-type C. elegans treated with daz-1 RNAi. (G) The fem-3(q20) mutant treated with daz-1 RNAi. Arrows indicate germ nuclei at the diakinesis stage during oogenesis. Arrowheads indicate the distal end of each gonad. Bar, 50 μm.

Figure 4.

Binding of the DAZ-1 protein to fbf mRNAs. (A) Expression pattern of DAZ-1-3xFLAG in the daz-1; btIs2 strain. Images of an extruded gonad from the daz-1; btIs2 strain, stained with either anti-FLAG antibody (top) or DAPI (bottom), are shown. Arrows indicate germ nuclei at the diakinesis stage during oogenesis. Arrowhead indicates the distal end of the gonad. Bar, 50 μm. (B) Coprecipitation of fbf mRNAs with DAZ-1. (a) Detection of DAZ-1-3xFLAG in the anti-FLAG immunoprecipitate from a btIs2 worm extract by immunoblotting. Tubulin is shown as a negative control. (b) Concentration of fbf-1 and fbf-2 mRNAs in the anti-FLAG immunoprecipitate, assayed by RT-PCR. A nonspecific binder rpl-1 is shown as a control. (C) Features of the 3′ UTR sequences of fbf-1 and fbf-2 mRNAs. (A/C/U)GUUC sequences are doubly underlined and U-rich tracts, singly. Sequences used as the RNA probes are shown in uppercase. Dotted nucleotides were replaced by the complementary ones in mutant competitors used in E. (D) Binding of DAZ-1 to the 3′ UTR of fbf mRNAs. The 3′ UTR fragment of fbf-1, fbf-2, or rpl-1 mRNA was biotin labeled, added to a btIs2 worm extract, and pulled down. DAZ-1-FLAG in the biotin-RNA precipitate was detected by immunoblotting using anti-FLAG antibody. S, sense-strand mRNA; AS, antisense-strand RNA. Tubulin is shown as a negative control. (E) In vitro reconstruction of DAZ-1-fbf mRNA complex. Gel retardation assay was performed to monitor the binding of GST-DAZ-1 protein (0–25 ng/μl) to ³²P-labeled sense strand 3′ UTR RNAs (2 ng/μl). Competitors are unlabeled sense RNA (13–100 ng/μl): F1, fbf-1; F2, fbf-2; F1mut, fbf-1 with GUnC mutated into CAnG; F2mut, fbf-2 with GUnC mutated into CAnG; and R, rpl-1.

Figure 5.

Reduction of the FBF protein in L4 larvae of the daz-1 mutant. FBF was detected by immunoblotting. One hundred and fifty hermaphrodites of the indicated genotype were collected at the L4 stage, boiled in SDS PAGE sample buffer, and loaded in one lane. Actin is shown as a loading control. The relative amount of FBF was quantified and indicated below each lane. (A) Wild type, daz-1(tj3), gld-3(ok308), and daz-1(tj3) gld-3(ok308) mutant at 20°C. (B) fem-3(q20) and daz-1(tj3); fem-3(q20) mutant at 25°C. (C) gld-1(q485); mock RNAi treatment and gld-1(q485); daz-1(RNAi) at 25°C.

Figure 6.

Compensation of the daz-1 mutation by the gld-3 mutation. (A–D) DAPI-stained images of extruded gonads from adult hermaphrodites. (A) Wild-type C. elegans. (B) The gld-3(ok308) mutant. (C) The daz-1(tj3) mutant. (D) The daz-1(tj3) gld-3(ok308) double mutant. Arrows indicate germ nuclei at the diakinesis stage during oogenesis, and arrowheads indicate the distal end of each gonad. (E–H) Magnified images of the proximal regions of the gonads shown in A–D, respectively, double stained with OMA-2 antibody (red) and DAPI (green). Bar, 50 μm.