A missense mutation in the Capza3 gene and disruption of F-actin organization in spermatids of repro32 infertile male mice

Abstract

Males homozygous for the repro32 ENU-induced mutation produced by the Reproductive Genomics program at The Jackson Laboratory are infertile, have low epididymal sperm concentrations, and produce sperm with abnormally shaped heads and poor motility. The purpose of the present study was to identify the mutated gene in repro32 mice and to define the structural and functional changes causing infertility and the aberrant sperm phenotype. In repro32/repro32 mice, we discovered a failure to shed excess cytoplasm and disorganization of the middle piece of the flagellum at spermiation, resulting in the outer dense fibers being wrapped around the sperm head within a bag of cytoplasm. Using a candidate-gene approach, a mutation was identified in the spermatid-specific "capping protein (actin filament) muscle Z-line, alpha 3" gene (Capza3). CAPZA3 protein localization was altered in spermatids concurrent with altered localization of a unique CAPZB variant isoform and disruption of the filamentous actin (F-actin) network. These observations strongly suggest the missense mutation in Capza3 is responsible for the mutant phenotype of repro32/repro32 sperm and regulation of F-actin dynamics by a spermatogenic cell-specific CAPZ heterodimer is essential for removal of the cytoplasm and maintenance of midpiece integrity during spermiation in the mouse.

Figure 1

Transmission electron microscopy (TEM) of the seminiferous epithelium and sperm in the cauda epididymis of repro32/repro32 and wild type mice. (A) Condensing spermatids (step 16) of repro32/repro32 and (B) wild type mice with the flagellum (fla) and cytoplasm (cyt) positioned for release from the seminiferous epithelium into the lumen of the tubule. (C) Sperm in the lumen of the cauda epididymis of repro32/repro32 and (D) wild type mice. Scale bar = 2 μ.

Figure 2

Cytoplasmic droplet (CD) localization in repro32/+ and repro32/repro32 sperm. (A, B) Differential interference contrast (DIC) images of repro32/+ sperm from the caput (A) and cauda (B) epididymis, with arrows indicating the locations of CDs. (C.i–iii) Examples of repro32/repro32 sperm from the cauda epididymis with their head and neck regions enclosed within a bag of cytoplasm (indicated by arrows). Scale bar = 5 μ.

Figure 3

Scanning electron microscopy (SEM) of cauda epididymal sperm from wild type and repro32/repro32 mice. (A) A wild type mouse sperm, showing the head, neck and proximal part of the middle piece (MP). (B) A repro32/repro32 mouse sperm with the head and MP enclosed within a bag of cytoplasm and the principal piece (PP) of the flagellum visible. (C) A wild type mouse sperm demembranated by detergent treatment. (D) A repro32/repro32 demembranated mouse sperm, showing the outer dense fibers (ODFs) and the middle piece wrapped around the sperm head. Scale bar = 2 μ.

Figure 4

A missense mutation (M44K) is present in the Capza3 gene in repro32/repro32 mice. (A) Partial mouse Capza3 coding sequence (nt 121-141, GenBank sequence NM_007605) from parental strains C3H and B6 and mutant strain repro32/repro32 mice. A T/A transversion changes an ATG to an AAG codon (dashed box). (B) A region of the CAPZA3 sequence common to the species indicated. The resultant missense mutation replaces a methionine (M, red box) with a lysine at position 44.

Figure 5

CAPZA3 localization and levels in spermatids. IIF with antiserum to CAPZA3 (green) on repro32/+ round (A.i), elongating (A.ii), early condensing (A.iii), and late condensing (A.iv) spermatids. CAPZA3 localization in repro32/repro32 round (B.i), elongating (B.ii) and condensing (B.iii–vi) spermatids. Upper panels = merged CAPZA3 IIF (green), DAPI (blue) and DIC images, lower panels = CAPZA3 IIF only. Scale bars = 2μ. (C) Immunoblot of the insoluble fraction of whole testis lysates from wild type and two repro32/repro32 mice. ACTB = cytoplasmic beta actin (loading control).

Figure 6

Genomic organization of a testis-specific Capzb transcript variant (Capzb_v3) and expression of Capzb transcript variants (Capzb_v1, Capzb_v2, and Capzb_v3) in the mouse. (A) The testis-specific Capzb_v3 transcript contains a unique N-terminal extension resulting from utilization of alternate exon 1b. Capzb_v1 and Capzb_v2 utilize exon 1a, but Capzb_v2 differs from Capzb_v1 due to alternative splicing that omits 113 nucleotides within exon 9. (B) RT-PCR using Capzb variant-specific primers (locations shown in A, upper F/R, lower F/R) to amplify 50 ng of cDNA from various tissues (NT = no template control, 0 ->60 dpp = whole testis aged in days post partum; P = pachytene spermatocytes, RT = round spermatids, Ser = Sertoli cells, Sk = skeletal muscle, Kid = kidney, Br = whole brain). Actb = cytoplasmic beta actin. (C) Testicular sperm from repro32/+ mice; (C.i) merged CAPZA3 IIF (green) and DAPI (blue); (C.iii) CAPZA3 IIF only; (C.ii) merged CAPZB IIF and DAPI; (C.iv) CAPZB3 IIF only. Scale bars = 2μ

Figure 7

Mechanically isolated spermatids from repro32/+ and repro32/repro32 mice with associated ES-containing Sertoli cell remnants. The F-actin fibers of the ES are labeled with fluorescent dye-conjugated phalloidin (red). (A) repro32/+ spermatid labeled with phalloidin and DAPI; (A′) enlarged image of same repro32/+ spermatid labeled with phalloidin alone; (B) repro32/repro32 spermatid stained with phalloidin and DAPI; (B′) enlarged image of same repro32/repro32 labeled with phalloidin alone. Cytoplasmic remnants are indicated by yellow arrows. Scale bars = 2μ.

Figure 8

Sertoli cell ESs have been removed from developing spermatids by trypsin treatment. (A) F-actin labeled with fluorescent dye-conjugated phalloidin (red) in repro32/+ (A.i) step 8 round, (A.ii) elongating, (A.iii) early condensing, and (A.iv) late condensing spermatids. F-actin was not detected in testicular sperm (A.v). (B) F-actin fiber development in repro32/repro32 (B.i) step 8 round, (B.ii) elongating, and (B.iii–vi) condensing spermatids. The upper panels of figures A and B are merged images of DIC, phalloidin-labeled F-actin (red), and DAPI-labeled nuclei (blue), while the bottom panels show phalloidin-labeled F-actin only. Yellow arrows indicate associated cytoplasm. Scale bars = 2μ.