Loss of polyadenylation protein τCstF-64 causes spermatogenic defects and male infertility

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   Fig. 1.

   Summary of defects in Cstf2t ^−/− mouse testes. Indicated is the timeline of mouse spermatogenesis (≈34 days), significant stages of spermatogenesis, and cell types in which CstF-64 (solid gray bar) and τCstF-64 (dashed box) proteins are expressed in wild-type mice. The first visible lesion in stage-XII secondary spermatocytes (arrow) and cumulative histological defects (step-10–16 spermatids, arrows and triangle) are indicated (see Fig. 3).

   
   
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   Fig. 2.

   Targeted disruption of Cstf2t eliminates expression of τCstF-64 in testes of Cstf2t ^−/− mice. (A) Targeted replacement of Cstf2t (top line) by a gene-encoding resistance to neomycin (NEO) (bottom line) using homologous recombination in 129SvEv mouse embryonic stem cells. (B) Genotyping mice that are wild-type (lane 1, +/+), heterozygous (lane 2, +/−), or homozygous (lane 3, −/−) for the Cstf2t^tm1(Neo) allele by genomic PCR. Sizes of fragments from the wild type (Cstf2t, 1,850 bp) or mutant (Neo, 1,306 bp) are indicated at the right. (C) Expression of τCstF-64 and CstF-64 mRNAs in wild-type (lane 1, +/+), heterozygous mutant (lane 2, +/−), or homozygous mutant (lane 3, −/−) Cstf2t mouse testes. Shown is ethidium bromide-stained agarose gel analysis of RT-PCR products from testes of wild-type (lane 1, +/+), heterozygous mutant (lane 2, +/−), or homozygous mutant (lane 3, −/−) Cstf2t mice; lane 4 (−RT) is RT-PCR performed but with reverse transcriptase omitted during the cDNA preparation step. Primers pairs were designed to detect τCstF-64 (366 bp) (Top), CstF-64 (256 bp) (Middle), or ribosomal S16 (382 bp) (Bottom) mRNAs. (D) Protein immunoblots of testis extracts using antibodies that recognize τCstF-64 [arrow, 70 kDa (Upper), 40 μg of protein per lane] or CstF-64 [arrow, 64 kDa (Lower), 20 μg of protein per lane] and α-actin [arrowhead, 43 kDa (Upper and Lower)]. Testis extracts (5) were from either wild-type (lane 1, +/+), heterozygous mutant (lane 2, +/−), or homozygous mutant (lane 3, −/−) Cstf2t mice.

   
   
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   Fig. 3.

   Lesions in Cstf2t ^−/− mouse testes. (A–C) Wild-type testes. Most tubules were normal in appearance, and stages of spermatogenesis consisted of the correct cellular associations. (A) Wild-type stage VII–VIII, consisting of step-7–8 and step-16 elongate spermatids (near lumen). (B) Wild-type stage XI, with step-11 elongating spermatids. (C) Wild-type stage XII with spermatocytes exhibiting condensed chromatin of meiosis 2 and step-12 spermatids. (D–I) Cstf2t ^−/− testes. (D) Cstf2t ^−/− testis in stage IX, with normal step-9 spermatids but with abnormal retention of step-16 spermatids (arrowheads), indicating failed spermiation. Large abnormal aggregates of residual bodies are seen attached to the retained spermatids (arrows). (E) Cstf2t ^−/− testis showing normal step-8 spermatids along with abnormal step-16 spermatids that are not aligning properly for spermiation (arrowheads). Abnormal residual bodies form near the lumen (arrows). (F) Cstf2t ^−/− stage XII containing several abnormalities, including degenerative spermatocytes in meiosis 2 (arrows, with PAS+ granulation), binucleate step-1 spermatids (circles), and abnormal elongated spermatid heads (arrowheads). (G) Cstf2t ^−/− stage I with normal step-1 spermatids, numerous abnormally shaped step-13 elongated spermatid heads (arrowheads) and evidence of sloughing of round spermatids (arrows). (H) Cstf2t ^−/− stage IX, with normal step-9 spermatids but showing abnormal step-16 spermatid heads (arrowheads) being retained within the epithelium. (I) Cstf2t ^−/− stage X, with some normal elongating spermatids (10) but also showing early formation of misshapen spermatid heads (arrowheads). Pachytene spermatocytes are also seen near the lumen, where they may be sloughed (arrow). (Scale bar: A–I, 25 μm.) (J) Cstf2t ^−/− mouse testis at lower magnification to show round spermatids (arrowheads), spermatocytes (arrows), and residual body debris (arrows) being sloughed into the lumen. (Scale bar, 50 μm.) (K) Cstf2t ^−/− mouse epididymis showing evidence of extensive sloughing of germ cells and debris by the testis (arrowheads).

   
   
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   Fig. 4.

   Microarray analyses. (A) Differentially expressed genes between wild-type and Cstf2t ^−/− mouse testes. SAM plots are shown for various time points of development (17, 22, and 25 dpp). Each dot is a probe set corresponding to differentially expressed genes, with red dots being up-regulated and green dots being down-regulated genes in knockout samples. (B) Venn diagram showing relationships of significantly changed genes in 22 and 25 dpp samples. (C) Hierarchical clustering performed using 8,913 probe sets selected by SAM and constructed by Pearson correlation and average linkage. Expression values for each probe set across all samples were median-centered and normalized, with red indicating values above and green indicating values below the median.