Widespread presence of human BOULE homologs among animals and conservation of their ancient reproductive function

Abstract

Sex-specific traits that lead to the production of dimorphic gametes, sperm in males and eggs in females, are fundamental for sexual reproduction and accordingly widespread among animals. Yet the sex-biased genes that underlie these sex-specific traits are under strong selective pressure, and as a result of adaptive evolution they often become divergent. Indeed out of hundreds of male or female fertility genes identified in diverse organisms, only a very small number of them are implicated specifically in reproduction in more than one lineage. Few genes have exhibited a sex-biased, reproductive-specific requirement beyond a given phylum, raising the question of whether any sex-specific gametogenesis factors could be conserved and whether gametogenesis might have evolved multiple times. Here we describe a metazoan origin of a conserved human reproductive protein, BOULE, and its prevalence from primitive basal metazoans to chordates. We found that BOULE homologs are present in the genomes of representative species of each of the major lineages of metazoans and exhibit reproductive-specific expression in all species examined, with a preponderance of male-biased expression. Examination of Boule evolution within insect and mammalian lineages revealed little evidence for accelerated evolution, unlike most reproductive genes. Instead, purifying selection was the major force behind Boule evolution. Furthermore, loss of function of mammalian Boule resulted in male-specific infertility and a global arrest of sperm development remarkably similar to the phenotype in an insect boule mutation. This work demonstrates the conservation of a reproductive protein throughout eumetazoa, its predominant testis-biased expression in diverse bilaterian species, and conservation of a male gametogenic requirement in mice. This shows an ancient gametogenesis requirement for Boule among Bilateria and supports a model of a common origin of spermatogenesis.

Figure 1. Phylogenetic distribution of motile sperm, Boule and Dazl homologs among species from major lineages of the animal kingdom and fungi.

Sexual reproduction in which male animals produce motile sperm often with flagellum is found in all major phyla of metazoan animals . Representative species of each major metazoan taxon (mostly phylum) were analyzed for the presence or absence of Boule and Dazl homologs. The common names for those species are listed with the names of the phyla or taxonomical groups they represent printed above their branches. Major superphyla groups and Chordata phylum are labeled vertically near the roots of their phylogenetic branches and bilaterian lineages (Bilateria) are highlighted in yellow. LTZ – Lophotrochozoa, ESZ – Ecdysozoa, Trout—rainbow trout (Oncorhynchus mykiss), Shark—elephant shark (Callorhinchus milii). All other species names are listed in supplementary information. *The absence of a Boule homolog in sponge is tentative since it is based on the draft genome of Amphimedon queenslandica (http://www.jgi.doe.gov/sequencing/statusreporter/psr.php?projectid=16318).

Figure 2. Conservation and prevalence of Boule proteins among animals.

(A) Alignment of Boule RRM domains from representative species of both deuterostomes and protostomes reveals conservation of the RNA binding domain, in particular in the regions surrounding RNP2 and RNP1 as well as the C-terminal region. Asterisks (*) mark amino acids conserved in Boule homologs from all species. The red bracket on the right indicates deuterostomian species and the green bracket protostomian species. Colors for individual amino acids are assigned based on similar properties of residues (Table S5). (B) Phylogenetic treatment of homologs from major metazoan branches of human DAZ gene family members (BOULE, DAZL and DAZ) using conserved RRM domains. Consistent with being the most ancient member of the DAZ family, the Boule clade is much more widespread and divergent, including members of the major phyla of protostomes and deuterostomes, while all DAZ/DAZL homologs are clustered together in one branch. The Dazl homolog clad and DAZ clad are much smaller and are restricted to vertebrates or primates, respectively. (C) Rooted tree showing the evolutionary distance among different homologs of the DAZ family. The numbers indicate bootstrap values. The evolutionary tree is drawn to scale, with branch lengths in units representing the number of amino acids substituted per site. Hs-Homo sapiens, Mm-Mus musculus, Md-Monodelphis domestica, Gg-Gallus gallus, Pm-Petromyzon marinus, Dl-Dicentrarchus labrax, Bf-Branchiostoma floridae, Hr-Helobdella robusta, Sm-Schistosoma mansoni, Bg-Biomphalaria glabrata, Dm-D. melanogaster, Sp-Strongylocentrotus purpuratus, Ce-C. elegans, Dr-Danio rerio, Xl-Xenopus laevis, Nv-Nematostella vectensis.

Figure 3. Conservation of reproduction-specific and testis-enriched expression of three deuterostome Boule homologs.

(A) RT-PCR survey using chicken Boule and Dazl specific primers on different tissues from adult rooster and hen (Gallus gallus). Dmrt1 (Doublesex and mab-3 related transcription factor 1) is a testis-specific gene and actin was used as a loading control. (B) RT-PCR survey using sea urchin-specific primers on different tissues from purple sea urchin (Strongylocentrotus purpuratus). Bnd (Bindin) is a testis-specific gene and ubiquitin was used as a loading control. W, water; Mu, muscle; Ki, kidney; He, heart; Br, brain; Li, liver; Ov, ovary; Te, testis; Am, Ampullae; Gu, guts. (C) Mouse Boule consists of 13 exons that encode multiple isoforms resulting from 5′ and 3′ alternative splicing. Two predominant mouse Boule isoforms – Bol1 and Bol2 – are male-biased and are restricted to or enriched in adult testes. Bol1 contains all 12 exons whereas Bol2 contains all exons except exon 11. RT-PCR analysis of RNA from mouse embryonic and adult gonads using primers corresponding to either exons 3 and 9 (upper panel, 501-bp band) or exons 3 and 12 (lower panel, 847-bp and 712-bp bands) reveals that full-length Bol1 is only expressed in postnatal testes, while Bol2 lacking exon 11 is also detectable at low levels throughout gonadal development in addition to its high adult testis expression. A similar pattern was confirmed using primers for exons 1 and exon 12.

Figure 4. Generation of a loss-of-function Boule mutation in mice.

(A) Gene targeting strategy with wild type genomic structure from exon 2 to exon 4 (top) and the successfully targeted locus (bottom). Thick bars represent the two genomic regions used for homologous recombination in the gene targeting construct. PvulI (P) restriction sites and the position of the 5′ probe are shown. (B) Southern hybridization with the 5′ probe shows that the wild type contains a 16-kb PvuII fragment, whereas homozygotes contain a 14-kb PvuII fragment and heterozygotes contain both 16-kb and 14-kb fragments, thus confirming homologous recombination. (C) Northern hybridization using Boule cDNA shows that all three wild type Boule transcripts are absent in the homozygotes; instead a novel transcript corresponding to exon 1, exon 2, and lacZ was detected. (D) RT-PCR using primers spanning exons 2 to 4 further confirmed the complete absence of wild type transcripts. Expected wild type products between exon 2 and 4 were present in heterozygotes and the wild type but absent in homozygotes. The weak amplification of a very large fragment in homozygotes represents the PCR product from the chimerical transcript spanning exon 2, LacZ, and exon 4 of the mutant allele.

Figure 5. Mouse Boule mutant males are infertile, resulting from a global arrest of spermatogenesis.

(A) Knockout mice are indistinguishable from those of wild type in morphology and size. (B) The wild type male reproductive tract consists of a pair of oval-shape testes (white arrow head) attached to the epididymis (white arrow), seminal vesicle (yellow arrowhead), and urethra (yellow arrow). (C) The reproductive tract in mouse Boule mutants appears normal, similar to that of wild type mice. Wild type epididymis tubules contain lots of motile sperm (D), and condensed sperm nuclei and sperm tails (arrow) are clearly visible (F at higher magnification). In the Boule mutant, epididymal tubules lack mature sperm and some are half-empty (E). Instead, small degenerating round cells are found inside the tubules (arrow in G). (H) Testicular sections from wild type mice showing the presence of sperm in the lumen (arrow in H) and elongating spermatids (arrowhead in J). (I, K) Sections from Boule mutant testes showing the absence of both sperm from the lumen (arrow in I) and elongating spermatids. Mouse Boule mutant testes contain degenerating cysts of germ cells (arrowheads in I and K). The scale bars are equal to 100 µm unless noted.

Figure 6. Schematic representation of evolution history of the three DAZ family members—Boule, Dazl, and DAZ—during metazoan evolution.

The birth of Boule is estimated to be after the divergence of placozoan from the common ancestor (open circle) of Cnidaria (such as sea anemone) and Bilateria. Dazl arose through duplication from ancestral Boule during the evolution of bony fish, possibly after its split from lamprey and cartilaginous fish but prior to the divergence of ray-finned fish and lobe-finned fish (i.e. tetrapod animal lineage). After their divergence from New World monkeys such as lemur, Old World monkey Dazl duplicated and gave rise to DAZ . Myr –millions of years. The geological scale is based on , .

Figure 7. Expression and functional requirement of Boule homologs in metazoan species studied.

The presence of a Boule homolog (+), testis expression or functional requirement (T) or ovary expression or functional requirement (O) is marked. Low level expression in the ovary is marked as a lower-case “o” to distinguish it from abundant ovarian expression, marked as a capital “O”. The information on Boule homologs are based on this work and previous reports , , , . The ancient Boule gene in the common ancestor of Bilateria (the Urbilaterian) is indicated as a filled circle, and ancestral Boule likely originated during the evolution of eumetazoans, shown as an open circle. The ancient bilaterian Boule was reproduction-specific, and we hypothesize that it functioned in spermatogenesis, based on a predominance of testis-biased expression in diverse lineages of bilaterian animals and the conservation of a male reproductive function in Drosophila and mouse.