key: cord-0705813-vw5p3l6t
authors: Yuan, Lihong; Zuo, Xueguo; He, Lingjiang; Racey, Paul; Levin, Eran; Zhang, Shuyi
title: Cloning and Characterization of Novel Isoforms of the BOULE Gene in Bats
date: 2009-11-28
journal: Biochem Genet
DOI: 10.1007/s10528-009-9299-2
sha: 067d52d080f7585520efbe856881f797682c1302
doc_id: 705813
cord_uid: vw5p3l6t

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Spermatogenesis is a complex physiological process controlled by many genes. The BOULE gene, a new member of the Deleted in Azoospermia (DAZ) family (which consists of BOULE, DAZ, and DAZ-like DAZl), is regarded as the ancestor of the DAZ family and a key factor in controlling the meiosis of male germ cells, which can regulate the expression of the twine gene and promote progression through meiosis (Eberhart et al. 1996; Karashima et al. 2000; Maines and Wasserman 1999; Xu et al. 2001) . Bats account for about 20% of mammals and, during evolution, have evolved many reproductive strategies, including sperm storage, delayed fertilization, delayed implantation, and delayed development (Nowak et al. 1994; Racey and Entwistle 2000) . The strategies of sperm storage and delayed fertilization allow many bat species, especially hibernating bats, to achieve synchrony between birth peaks and food availability (Racey 1979) . As little is known about the cell biology of bat spermatogenesis, in this study, we obtained BOULE gene sequences from four bat species (Rhinolophus ferrumequinum, Myotis ricketti, Eonycteris spelaea, and Rousettus leschenaultii) to test the role of the BOULE gene in bat spermatogenesis. We identified at least two isoforms (named a and b) of the BOULE gene in bats. Isoform a is common to the BOULE gene of other species, whereas isoform b, which is here identified for the first time, is specific in bats. As for the difference of exons lost, the bat BOULE gene isoform b has a premature stop codon in a different position and has different lengths of coding-domain sequence (CDS). Thus isoform b of the BOULE gene in some species may have lost the functional domain. Therefore, our study first cloned the multiple transcript variants of the bat BOULE gene and identified the novel isoforms. These results may add some useful information for the study of bat spermatogenesis and other reproductive strategies of bats.

One individual of each species (R. ferrumequinum and M. ricketti have sperm storage ability, E. spelaea and R. leschenaultii do not) was sacrificed as part of a surveillance program for coronaviruses in 2006, and testis tissue was stored at -80°C until RNA extraction. Total RNA was isolated from testes using RNAiso Reagent (TaKaRa, Japan), following the manufacturer's protocol. The concentration of RNA was calculated according to the formula [RNA] = 50 9 (OD 260 -OD 320 ) 9 dilution time, and the quality of total RNA was assessed on a 1% agarose gel.

cDNA Synthesis and RT-PCR Amplification Two micrograms of total RNA from each sample were used to synthesize cDNA. First, 2 lg total RNA was treated with 2 U RNase-free DNase I (Promega) for 30 min at 37°C to avoid genomic DNA contamination, then converted to cDNA by Superscript III Reverse Transcriptase (Invitrogen), according to the manufacturer's instructions, in a 50 ll reaction mixture containing 500 ng random primer, 1 mM dNTP, 2 mM dithiothreitol, 80 U RNase inhibitor (Promega), 19 First-Strand buffer, and 400 U Superscript III Reverse Transcriptase. The reaction conditions were 65°C for 5 min, followed by ice incubation for 5 min, then 25°C for 5 min, 50°C for 1 h, and finally 70°C for 15 min.

The degenerated primer pairs PF1 (5 0 GRC GCA ARC AWC AAA YCA GAT GCA AAC AGA 3 0 ) and PR1 (5 0 AGY TGG AMT AGA GCT GCC CAA TTG TCT TAA3 0 ), according to the conservative part of the nucleotide sequences of the BOULE gene from several mammals, were synthesized to obtain the completed CDS of the bat BOULE gene. Using the first-strand cDNAs of bats as templates, PCR was performed at 94°C first predenatured for 5 min, 30 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, and finally 72°C extended for 10 min.

PCR products were isolated from 1% agarose gel and purified using the agarose gel DNA purification kit version 2.0 (TaKaRa, Japan), followed by ligation with the pGEM-T-easy vector (Promega, USA), then transformed into the DH5a competence cell (TaKaRa, Japan). The identity and orientation of each clone were verified by the universal M13 (-47)/(-48) primer, and then each was sequenced from both directions on an ABI 3730A automated DNA sequencer. To avoid artifacts, multiple clones were sequenced for every specimen.

Sequence Alignment and Structure Analysis of the BOULE Gene Fourteen BOULE CDS sequences, from Homo sapiens (NM_033030), Pan paniscus (AJ717405), Pan troglodytes (XM_516011), Callithrix jacchus (AJ717407), Saguinus oedipus (AJ717406), Saimiri sciureus (AJ717408), Macaca mulatta (XM_001086915), Macaca fascicularis (AB074454), Microcebus murinus (AJ746579), Canis familiaris (XM_545580), Bos taurus (NM_001102115), Equus caballus (XM_001500223), Rattus norvegicus (XM_001067043), and Mus musculus (AF272859), were obtained from GeneBank. The putative coding region of BOULE of Pongo pygmaeus was retrieved from the UCSC database (http://genome.ucsc.edu).

Nucleotide sequences were aligned using Clustal X 1.81 (Thompson et al. 1997 ) and used to generate amino acid alignments with Mega 4.0 (Tamura et al. 2007) .

A search for sequences on the NCBI database found eight clones of the BOULE gene from four bat species (GenBank acc. nos, FJ541190-FJ541197).

Sequence alignments indicated that there were at least two transcript variants for each bat BOULE gene, named isoform a and b. Further analysis showed that transcript variants of the bat BOULE gene differ mainly in the 3 0 end of their CDS, due to selective splicing (Table 1 ). The CDS of four bat species isoform a were encoded by 10 exons (a-j) and were identical to H. sapiens isoform 2. However, for E. spelaea isoform a, a 14 bp (AGGAGTGGGGAGTA) insert in exon i results in an open reading frame shift, leading to a premature stop of translation and a change of the amino acid sequence in exon i. Moreover, the CDS of M. ricketti and R. leschenaultii isoform b consists of nine exons, and the lack of exon i does not affect the open reading frame. Isoform b of R. ferrumequinum had three exons and E. spelaea had two exons. Exon d in R. ferrumequinum and exon b in E. spelaea are lost, their open reading frames are changed, and they have a stop codon in exon e and c, respectively (Fig. 1 ).

Gametogenesis involves specification of germ cell fate, mitotic replication of early germ cell populations, meiotic and postmeiotic development, and complex regulation at the levels of transcription and translation (Urano et al. 2005) . The BOULE gene family consists of three RNA-binding proteins, BOULE, Daz, and Daz-like (Dazl), that regulate germ cell development and differentiation (Houston and King 2000) . BOULE encodes a critical conserved switch that regulates progression of germ cells through meiosis in men (Xu et al. 2003) . The full-length of the human BOULE gene is 2046 bp, including a coding region (849 bp), an untranslated region (325 bp), introns (872 bp), and a DAZ repeat, and the human BOULE protein consists of 283 amino acids with a molecular weight of 31.3 kDa (Xu et al. 2003) . As an RNA-binding protein like other members of the DAZ family, BOULE comprises an RNA recognition motif (RRM) including two ribonucleoprotein signal motifs (RNP1 and RNP2) and a DAZ repeat (Reynolds and Cooke 2005) (Fig. 1) . The translational or transcriptional induction of BOULE required an RNA-binding protein or transcriptional factor. In Drosophila, BOULE is a post-transcriptional regulator of a CDC25 homolog called twine, which is required for the G2-M transition in the meiotic cell cycle during spermatogenesis (Eberhart et al. 1996) . Twine encodes CDC25-type phosphates and activates the maturation promoting factor (MPF), consisting of the cdc2/cyclinB complex (Sigrist et al. 1995) . So we hypothesize that the BOULE gene may participate in the regulation of spermatogenesis of bats. To test whether and how the BOULE gene plays a role in spermatogenesis, we cloned the BOULE gene from four bat species, two of them with the ability to store sperm.

The human BOULE has three major species of transcripts (B1, B2, and B3), which differ only in their 5 0 ends, specifically in exon 1. Among these isoforms, B2 plays a major role in meiotic completion (Kostova et al. 2007) . Sequencing results showed that there were at least two transcript variants in each bat species. Isoform a The region required for homodimerization and interaction with the DAZ protein and PUM2 protein (Urano et al. 2005 ) is indicated by a line above the human sequence. Solid arrows indicate potential splice sites of the DAZ family can stimulate translation inhibition by interacting with poly(A)binding proteins (PABPs), and deletion of the RRM domain will completely abrogate the interaction with PABP (Collier et al. 2005) . Moreover, RRM and the DAZ repeat of BOULE are required for interaction with Pumilio-2 (PUM2). By binding with Pumilio, BOULE can relieve the repression of Pumilio on a B cyclin in order to promote meiotic G2/M translation (Urano et al. 2005) . Thus, the loss of RRM or the DAZ repeat of bat BOULE may disrupt its control of the transcription and translation of target genes, resulting in the progress of spermatogenesis being disrupted.

The multiple transcript isoforms of BOULE gene, including the lost functional domains suggest a new regulatory mechanism in bat spermatogenesis, and these sequences may aid in the understanding of the reproductive strategies of bats.

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Caenorhabditis elegans homologue of the human azoospermia factor DAZ is required for oogenesis but not for spermatogenesis

Association of three isoforms of the meiotic BOULE gene with spermatogenic failure in infertile men

Post-transcriptional regulation of the meiotic Cdc25 protein Twine by the Dazl orthologue Boule

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Role of the DAZ genes in male fertility

Dmcdc2 kinase is required for both meiotic divisions during Drosophila spermatogenesis and is activated by the Twine/cdc25 phosphatase

MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0

The Clustal X Windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools

Interaction of the conserved meiotic regulators, BOULE (BOL) and PUMILIO-2 (PUM2)

A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans

Human BOULE gene rescues meiotic defects in infertile flies