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First published online 17 December 2008
doi: 10.1242/dev.030858

Development 136, 373-382 (2009)
Published by The Company of Biologists 2009
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RIM-BP3 is a manchette-associated protein essential for spermiogenesis

Jing Zhou1,*, Ya-Rui Du1,*, Wei-Hua Qin1,{dagger}, Ye-Guang Hu1, Yan-Nv Huang1, Lan Bao2, Daishu Han3, Ahmed Mansouri4 and Guo-Liang Xu1,{ddagger}

1 The State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
2 The Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
3 Department of Cell Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, 5 Dong Dan San Tiao, Beijing 100005, China.
4 Max Planck Institute of Biophysical Chemistry, Department of Molecular Cell Biology, Am Fassberg, 37077 Goettingen, Germany.


Figure 1
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  		Fig. 1. Specific expression of RIM-BP3 in the mouse testis. (A) The RIM-BP3 protein showing the conserved domains and three antigen regions for raising antibodies. (B) Northern hybridization analysis of the RIM-BP3 transcript in different tissues as indicated above. The mRNA blot (Clontech) contains 2 µg mRNA on each lane. The hybridization probe corresponds to the C-terminal coding sequence (nucleotides 3547-4260). (C) Western analysis of the RIM-BP3 protein in different tissues as indicated above. The detection of {alpha}-tubulin serves as a loading control (lower panel). (D) Western detection of the RIM-BP3 protein in developing postnatal testis. Upper panel, diagram of stages of mouse spermatogenesis; PL, preleptotene spermatocytes; L, leptotene spermatocytes; Z, zygotene spermatocytes; P, pachytene spermatocytes; D, diplotene spermatocytes; MI, meiosis I; MII, meiosis II; middle panel, western analysis of RIM-BP3 (0-60 days post partum); bottom panel, Coomassie blue staining of the protein samples. (E) Western detection of RIM-BP3 in purified male germ cells. A, type A spermatogonia; B, type B spermatogonia; RS, round spermatids; E1 and E2, two pools of elongate spermatids; SP, mature sperm; RB, residual bodies; ST, Sertoli cells. Other abbreviations used are as in D. The lower panel shows Coomassie blue staining of samples used for the western analysis.

 

Figure 2
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  		Fig. 2. Distribution of the RIM-BP3 protein in elongating spermatids. Testicular germ cells were prepared from adult wild-type mice, and indirect immunofluorescence staining was performed using polyclonal anti-RIM-BP3 SbcC (green) and monoclonal anti-{alpha}-tubulin (red) antibodies. The slides were counterstained with DAPI to visualize the nucleus. Testicular germ cells from RIM-BP3 knockout mice were used as negative controls (see Fig. S2 in the supplementary material). Scale bar: 10 µm.

 

Figure 3
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  		Fig. 3. Targeted disruption of the RIM-BP3 gene. (A) The strategy for the generation of a targeted RIM-BP3 allele. Black bars represent coding regions of the single-exon gene. BamHI restriction sites (B) and the probe used for Southern analysis (R), and PCR primers (arrows) used for genotyping are indicated. (B) Genotype confirmation of the knockout mouse by PCR (upper panel) and Southern analysis (lower panel). The DNA fragments derived from the wild-type (WT) and mutant (MT) alleles are indicated on the right. +/+, wild type; +/-, heterozygote; -/-, homozygote. (C) Genotype confirmation of the knockout mouse by western analysis. The blot containing testis protein extracts from wild-type (+/+), heterozygous (+/-) and homozygous (-/-) adult mice was probed with antibodies specific for N- and C-terminal regions (Fig. 1A). The detection of β-actin serves as a loading control.

 

Figure 4
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  		Fig. 4. Morphological abnormalities of epididymal sperm from RIM-BP3 knockout mice. (A) Scanning electron micrographs. (a) Abnormal head shaping, tail detachment (arrowheads) and bending (arrow) occur in sperm of the mutant mouse. Scale bar: 20 µm. (b,c) Higher magnification of abnormal spermatid heads lacking the hook-shaped appearance typical of a normal spermatid from the wild-type mouse (d). Scale bar in d: 2 µm in b-d. (B) Transmission electron micrographs. Deformed nuclei were observed (asterisk) and most of the sperm heads in mutant mice display a detached acrosome (black arrow). Acr, acrosome; Nu, nucleus. Scale bars: 1 µm.

 

Figure 5
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  		Fig. 5. Spermiogenesis abnormalities in RIM-BP3 knockout mice revealed by transmission electron microscopy. (A,B) Step 5 spermatids. Unfused proacrosomal vesicles are aligned above the bent acroplaxome in a mutant round spermatid (B). The ends of the extending acroplaxome in this section are marked with white arrows. (C,D) Step 9 spermatids. Acrosome fragmentation was occasionally observed in mutant elongating spermatids as shown in D. The breakage point is indicated with an arrowhead (D, inset). (E-G) Step 10/11 spermatids. At this elongating stage, abnormalities ranged from acrosome discontinuity (F, inset, arrowhead), acroplaxome distortion (F,G) and nuclear shape irregularity to symmetric manchette appearance with a conical shape (F). Black arrows indicate the perinuclear ring of the machette. (H,I) Step 12/13 spermatids. A membrane-bound microtubular bundle is ectopically located within the nuclear envelope on the presented cross-section (H, inset). The perinuclear ring (I, left arrow) at the left is shifted away from its normal location subjacent to the marginal ring (compare with the perinuclear ring on the right, arrow). (J-L) Step 15/16 spermatids. The mutant elongated spermatids frequently contain deformed nucleus and detached acrosome, with some unidentified materials in the expanded perinuclear space. Acrosome gaps (arrowheads, K) continue to exist. Nu, nucleus; Acr, acrosome; M, manchette. Scale bars: 1 µm.

 

Figure 6
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  		Fig. 6. The RIM-BP3 protein is associated with Hook1. (A) Purification and identification of proteins associated with RIM-BP3. Protein extracts of spermatids isolated from wild-type (WT) mice and RIM-BP3 knockout (KO) mice were immunoprecipitated with anti-RIM-BP3 (N) or anti-RIM-BP3 (C) antibody. The immunoprecipitated proteins were separated by SDS-PAGE and visualized by silver staining. The prominent protein specifically co-purified with RIM-BP3 was identified as Hook1 by mass spectrometric analysis. The detected Hook1 peptides are listed in the box, with their amino acid positions indicated. (B) Co-immunoprecipitation of endogenous RIM-BP3 and Hook1. The total testicular extracts from RIM-BP3 knockout (lane 3) and wild-type mice (lane 4) were immunoprecipitated with the anti-RIM-BP3 (C) antibody. The immunoprecipitates were fractionated by SDS-PAGE and blotted with anti-Hook1 antibody. Left two lanes are 0.6% of the input extracts used for immunoprecipitation. (C) Mapping of the interaction region in RIM-BP3 by yeast two-hybrid assay. RIM-BP3 full-length and its four fragments (upper panel) were fused to GAL4AD and co-transformed into yeast with the bait construct GAL4BD-Hook1. The interaction capability of each fragment was judged by the appearance of colonies on the SD minimal medium (SD-L-T-A-H) (lower panel). (D) Mapping of the interaction region in Hook1 by yeast two-hybrid assay. Full-length Hook1 and its three fragments (upper panel) were fused to GAL4BD and co-transformed into yeast with the prey construct GAL4AD-RIM-BP3. Growth of colonies on the SD minimal medium (SD-L-T-A-H) reflects positive interaction (lower panel). MT, microtubule-binding domain; O-binding, organelle-binding domain (Mendoza-Lujambio et al., 2002Go).

 

Figure 7
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  		Fig. 7. The Hook1 azh mutant is unable to interact with RIM-BP3. (A) Schematic representation of the wild-type Hook1 and the azh mutant Hook1 protein (Hook1azh) with their polypeptide length indicated. (B) The expression constructs of Flag-RIM-BP3, HA-Hook1 and HA-Hook1azh were co-transfected into HEK-293T cells with various combinations indicated at the top. Whole-cell extracts were immunoprecipitated with the anti-RIM-BP3 (C) or anti-HA antibody, and the precipitated proteins were analyzed by western blotting using anti-HA or anti-RIM-BP3 (C) antibody as indicated.

 

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