A gene trap knockout of the abundant sperm tail protein, outer dense fiber 2, results in preimplantation lethality

Abstract

Outer dense fiber 2 (Odf2) is highly expressed in the testis where it encodes a major component of the outer dense fibers of the sperm flagellum. Furthermore, ODF2 protein has recently been identified as a widespread centrosomal protein. While the expression of Odf2 highlighted a potential role for this gene in male germ cell development and centrosome function, the in vivo function of Odf2 was not known. We have generated Odf2 knockout mice using an Odf2 gene trapped embryonic stem cell (ESC) line. Insertion of a gene trap vector into exon 9 resulted in a gene that encodes a severely truncated protein lacking a large portion of its predicted coil forming domains as well as both leucine zipper motifs that are required for protein-protein interactions with ODF1, another major component of the outer dense fibers. Although wild-type and heterozygous mice were recovered, no mice homozygous for the Odf2 gene trap insertion were recovered in an extended breeding program. Furthermore, no homozygous embryos were found at the blastocyst stage of embryonic development, implying a critical pre-implantation role for Odf2. We show that Odf2 is expressed widely in adults and is also expressed in the blastocyst stage of preimplantation development. These findings are in contrast with early studies reporting Odf2 expression as testis specific and suggest that embryonic Odf2 expression plays a critical role during preimplantation development in mice.

FIG. 1

Characterization of Odf2 knockout allele. (a) Long range PCR mapping between exons 8 and 9 of the Odf2 gene. (b) Sequence of Odf2 exon 9 from the wild-type and mutant allele; italics represent vector sequence and underlining denotes additional sequence at insertion site. (c) Schematic showing vector insertion site in exon 9 and location of LacZ probe used for Southern blotting; vector sequence is represented by the dashed line and the β-geo gene is represented by the dotted box. (d) PCR genotyping strategy differentiates between wild-type (147) bp and mutant (676) bp bands. (e) Southern blotting with the LacZ probe identifies 3.1 kb vector band.

FIG. 2

Characterization of Odf2 mutant mRNA transcript. (a) RT-PCR from adult testis RNA. Primers amplified specific regions either upstream of the insertion site (Up) or across the insertion site from wild-type to vector sequence (Vector). (b) Sequence of Odf2 exon 9 showing the cryptic splicing site used in the mutant, relative to the insertion site of the vector. (c) Northern blot of Odf2 mRNA isolated from adult testis, probed with Odf2, β-geo, and Gapdh probes. (d) Quantitative RT-PCR of Odf2 mRNA isolated from adult testes isolated from wild-type (WT) and heterozygous (Het) mice. Expression is normalized to Gapdh. Data is presented as mean ± SEM; bars with different letters are significantly different (P > 0.05).

FIG. 3

Odf2 mRNA expression in wild-type adult tissues and blastocysts, and ODF2: LACZ expression in the heterozygous testis. (a) RT-PCR of Odf2 and Gapdh from total RNA isolated from adult wild-type mice: Br = brain, Ep = epididymis, He = heart, Ki = kidney, Li = liver, Lu = lung, Ov = ovary, Te = testis, St = stomach, and Ut = uterus. (b) RT-PCR of Odf2 and Gapdh from total RNA isolated from blastocysts; H₂0 was included as a negative control and testis cDNA was included as a positive control. (c–f) ODF2:LACZ expression within testis sections from adult mice heterozygous for the Odf2 insertion: (c) Seminiferous tubules viewed at ×100; (d) Elongating spermatozoa viewed at ×1,000; (e) Spermatozoa in the seminiferous tubule viewed at ×1,000; (f) Caput epididymis viewed at ×100.

FIG. 4

Sperm counts and motility assays. (a) Sperm counts from the entire epididymide of adult wild-type (WT) and Odf2 heterozygous (Het) mice. (b) Sperm motility assays from the caudal epididymide of adult wild-type (WT) and heterozygous (Het) mice. Sperm progressing across the slide were recorded as ‘progressing’ while ‘motile’ refers to total sperm showing signs of motility, from repeated twitching to progression.