Abnormal sperm in mice lacking the Taf7l gene

Abstract

TFIID is a general transcription factor required for transcription of most protein-coding genes by RNA polymerase II. TAF7L is an X-linked germ cell-specific paralogue of TAF7, which is a generally expressed component of TFIID. Here, we report the generation of Taf7l mutant mice by homologous recombination in embryonic stem cells by using the Cre-loxP strategy. While spermatogenesis was completed in Taf7l(-/Y) mice, the weight of Taf7l(-/Y) testis decreased and the amount of sperm in the epididymides was sharply reduced. Mutant epididymal sperm exhibited abnormal morphology, including folded tails. Sperm motility was significantly reduced, and Taf7l(-/Y) males were fertile with reduced litter size. Microarray profiling revealed that the abundance of six gene transcripts (including Fscn1) in Taf7l(-/Y) testes decreased more than twofold. In particular, FSCN1 is an F-action-bundling protein and thus may be critical for normal sperm morphology and sperm motility. Although deficiency of Taf7l may be compensated in part by Taf7, Taf7l has apparently evolved new specialized functions in the gene-selective transcription in male germ cell differentiation. Our mouse studies suggest that mutations in the human TAF7L gene might be implicated in X-linked oligozoospermia in men.

FIG. 1.

Contrasting exon/intron structures and expression patterns of the mouse Taf7l and Taf7 genes. (A) Taf7 is a retroposed derivative of Taf7l. The gene structures were determined by alignment of Taf7l (accession no. AK017109) and Taf7 (NM_011901) cDNA sequences with their genomic sequences. Coding regions are shown in black. Percent identities in the coding regions for nucleotide (nt) and aa sequences are indicated. Compared with TAF7, TAF7L contains ∼100 additional residues at its amino terminus. No significant nt identity is present in the untranslated regions (UTR). (B) Western blot of TAF7L and TAF7 in adult mouse tissues. Equal amounts (20 μg) of protein extracts for each tissue were loaded. β-Actin served as a control. Chr., chromosome.

FIG. 2.

Targeted inactivation of Taf7l in mice. (A) Schematic presentation of the Taf7l gene, the targeting construct, and various alleles. Exons 1 to 9 are shown as rectangles. Exons 10 to 13 are not shown. Deletion of exons 2 to 6 (aa 96 to 263) in the Taf7l mutant allele is expected to cause a frameshift. (B) Genotyping of Taf7l alleles. Genotypes are indicated. wt, wild-type allele; mt, Taf7l mutant allele. (C) Western blot of Taf7l^−/Y testes. Equal amounts (20 μg) of testis protein extracts were loaded. TAF7L was absent in Taf7l^−/Y testes. The abundances of TAF7 and TAF4 did not differ in Taf7l^−/Y and wild-type testes. (D) Histological analysis of 7-month-old Taf7l^−/Y testes. Two large vacuoles (arrows) were present in the seminiferous tubule, despite the presence of a full spectrum of germ cells.

FIG. 3.

Competition between TAF7 and TAF7L for association with TBP. (A) Expression analysis of TBP and TBP-associated factors in Taf7l^−/Y testes. Ten micrograms of two independent extracts from testes of wild-type (Taf7l^+/Y) or Taf7l^−/Y animals was probed with antibodies against the indicated proteins. The filter was then reprobed with antibodies against TAF5 and TAF6. (B) Results of coimmunoprecipitation assays. Lanes 1 and 2 show starting extracts from wild-type and mutant testes used for immunoprecipitation (IP) with anti-TBP antibody. Lanes 3 and 4 show peptide-eluted material from immunoprecipitations. The filter was probed with antibodies against TBP, TAF7, and TAF7L and then reprobed with antibodies against TAF6. To avoid masking TAF7 with a signal from the heavy chain of anti-TBP antibody used in immunoprecipitation, the blot was revealed using conjugated goat anti-mouse κ chain antibody as previously described (24).

FIG. 4.

Morphological defects in Taf7l-deficient sperm. Sperm from adult cauda epididymides were analyzed. (A) Wild-type sperm. (B) Taf7l mutant sperm folded at the proximal middle piece. The sperm head is bent back on the tail. (C) Flagellar angulation of Taf7l mutant sperm at the distal middle piece. (D) The middle piece is bent over the principal piece. In addition, the principal piece of Taf7l mutant sperm is abnormally curved. Arrows indicate a junction between the middle and principal pieces. (E) Percentage of sperm with normal and angulated tails. Three 8-week-old mice of each genotype (Taf7l^+/Y or Taf7l^−/Y) were analyzed. Two hundred sperm from cauda epididymides were counted for each animal.

FIG. 5.

Ultrastructural defects in Taf7l-deficient sperm. (A) Wild-type sperm. (B) Taf7l mutant sperm. The sperm head (n) is folded over the middle piece (mp). Arrows indicate the continuity of the cytoplasmic membrane from the apex of the sperm head to the middle piece. (C) Taf7l mutant sperm. The principal piece (pp) is bent 180° over the middle piece around the annulus. Bar, 500 nm.

FIG. 6.

Expression analysis during spermatogenesis. Relative transcript levels among different spermatogenic cell populations were assayed by RT-PCR. Actb served as a ubiquitous expression control. Pgk2 is transcribed at the onset of meiosis. Prm1 is expressed in postmeiotic germ cells. A, type A spermatogonia; B, type B spermatogonia; PL, preleptotene spermatocytes; LZ, mixed leptotene and zygotene spermatocytes; PS, pachytene spermatocytes; RS, round spermatids; WT, wild-type adult testes; XX^Y*, germ cell-deficient adult testes.