Ovol2, a mammalian homolog of Drosophila ovo: gene structure, chromosomal mapping, and aberrant expression in blind-sterile mice

Abstract

The ovo gene family consists of evolutionarily conserved genes including those cloned from Caenorhabditis elegans, Drosophila melanogaster, mouse, and human. Here we report the isolation and characterization of mouse Ovol2 (also known as movol2 or movo2) and provide evidence supporting the existence of multiple Ovol2 transcripts. These transcripts are produced by alternative promoter usage and alternative splicing and encode long and short OVOL2 protein isoforms, whose sequences differ from those previously reported. Mouse and human OVOL2 genes are expressed in overlapping tissues including testis, where Ovol2 expression is developmentally regulated and correlates with the meiotic/postmeiotic stages of spermatogenesis. Mouse Ovol2 maps to chromosome 2 in a region containing blind-sterile (bs), a spontaneous mutation that causes spermatogenic defects and germ cell loss. No mutation has been detected in the coding region of Ovol2 from bs mice, but Ovol2 transcription was dramatically reduced in testes from these mice, suggesting that Ovol2 is expressed in male germ cells.

FIG. 1

Analysis of mouse and human OVOL2 gene products. (A) The 5′ end sequences of the mouse Ovol2B cDNA and the deduced OVOL2B protein. The “#” symbol indicates the position of an internal methionine previously mistaken as the initiation codon [14]. (B) Deduced amino acid sequences of OVOL2A proteins in mouse (mOvol2A) and human (hOvol2A). The “*” symbol indicates amino acid identity. The four C2H2 zinc fingers are underlined. The predicted NLS sequences are boxed. Sequences common to mouse OVOL2A and OVOL2B start at the brackets in (A) and (B). Human OVOL2B starts at the internal methionine (bold). Shown in bold and italics are positions where our predicted sequence differs from the previously reported sequence [14]. (C) Phylogenetic analysis of OVO proteins. cOvo, C. elegans OVO (GenBank acc. no. AF134806); dOvo, Drosophila OVO (GenBank acc. no. X59772); mOvol1, mouse OVOL1 (GenBank acc. no. AF134804); hOvol1, human OVOL1 (GenBank acc. no. AF016045); mOvol2, mouse OVOL2 (GenBank acc. no. AY090537); hOvol2, human OVOL2 (GenBank acc. no. AK022284); mOvol3, mouse OVOL3 (GenBank acc. no. BF714064); hOvol3, human OVOL3 (GenBank acc. no. AD001527).

FIG. 2

Gene structure of mouse and human OVOL2. (A) Exon–intron structure of mouse Ovol2 (movol2). Rectangular boxes represent exons (striped, coding sequences; open, noncoding sequences) and horizontal lines represent introns. Only exons are drawn to scale. The filled circle denotes a zinc finger. Arrows indicate positions and directions of primers used in RT-PCR: a, E1A1; b, WYF1; c, WYB4; d, F2; e, R2. The 177-bp intron between E1B and E2 was mistakenly included in the mouse ovol2B cDNA sequence previously reported [14]. (B) RT-PCR analysis of Ovol2A transcripts from mouse testis. Lane 1, 100-bp size marker; lane 2, primers E1A1 and WYB4; lane 3, primers WYF1 and WYB4. (C) Exon–intron structure of human OVOL2 (hovol2).

FIG. 3

Tissue-specific expression patterns of OVOL2 in mouse and human. (A) Northern blot analysis of mouse tissue RNAs (Origene blots) using a probe containing sequences common to mouse Ovol2A and Ovol2B transcripts. (B) RT-PCR using primers F2 and R2 (Fig. 2A) common to mouse Ovol2A and Ovol2B transcripts (top), or using actin primers (bottom). (C) Northern blot analysis of human tissue RNAs (Clontech blots) using a probe containing sequences common to human Ovol2A and Ovol2B transcripts.

FIG. 4

Northern blot analysis of mouse Ovol2 expression in keratinocytes. Total RNAs were prepared from mouse keratinocytes cultured in low Ca²⁺ medium (-) or treated with Ca²⁺ for hours (hr) indicated. A probe detecting both mouse Ovol2A and Ovol2B transcripts (movol2) was used. GAPDH was used as a control for loading.

FIG. 5

Northern blot analysis of Ovol2 expression during prepubertal testis development. (A) Diagram of the process of spermatogenesis. In the seminiferous tubules of testis, some of the mitotically active germ cells (spermatogonia) enter the program of differentiation to yield primary spermatocytes (corresponding to meiotic prophase and subdivided into several stages including preleptotene, leptotene, zygotene, pachytene, and diplotene) [24]. Primary spermatocytes undergo meiosis I to yield secondary spermatocytes, which then undergo meiosis II to yield haploid spermatids. Spermatids subsequently undergo morphological transformations to produce mature spermatozoon. The timing of the first appearance of germ cells of different differentiation stages in the first round of spermatogenesis is shown on the right [adapted from 16]. (B) Mouse Ovol2 transcription in testis is upregulated at 3 weeks (w) after birth. (C) Abundant expression of mouse Ovol2A and Ovol2B transcripts correlates with the appearance of late meiotic/postmeiotic germ cells. Probes either common to mouse Ovol2A and Ovol2B transcripts (movol2A/B in B, C) or specific to each isoform (movol2A, movol2B in C) were used. Ages (weeks in B and days in C) at which testis samples were taken are shown at the top.

FIG. 6

Chromosomal mapping of mouse Ovol2 and characterization of Ovol2 sequence and expression in bs mice. (A) FISH analysis. Red dots (arrows) in the left panel represent hybridization signals obtained with a mouse Ovol2-containing BAC clone. On the right is the banding pattern of mouse chromosome 2. (B) Sequence analysis of mouse Ovol2 in bs mice. Mouse Ovol2 gene structure is shown at the top. Thick lines represent PCR fragments analyzed. For each fragment, at least two independent PCR reactions were carried out and the resulting fragments sequenced. (C) Northern blot analysis. Total testis RNAs were prepared from wild-type AKR mice (+/+), bs homozygous (bs/bs), and control littermates of bs/bs having either a +/+ or +/bs genotype.