TMEM95 is a sperm membrane protein essential for mammalian fertilization
- Animal Reproduction Department, INIA, Spain
- Department of Cell Biology and Histology, Medical School, University of Murcia, IMIB-Arrixaca, Spain
- Cell Surface Signalling Laboratory, Wellcome Trust Sanger Institute, United Kingdom
- School of Agriculture and Food Science, University College Dublin, Ireland
- Department of Biology, Faculty of Sciences, Institute of Food and Agricultural Technology, University of Girona, Spain
Figures
Figure 1 with 3 supplements
Generation of TMEM95 deficient mice.
(A) Structure prediction of TMEM95 protein (left) using IZUMO1 (right) as template, created by SWISS-MODEL software. (B) Tmem95 KO allele generated following CRISPR-mediated edition. CRISPR target …
Figure 1—figure supplement 1
Offtarget analysis.
None of the 5 most probable off-target sites for the sgRNA used were edited, showing the same sequence as the WT. Location of the possible off-target is coloured in gray.
Figure 1—figure supplement 2
WB images related to Figure 1D.
(A) Immunoblotting of protein extracts obtained from WT and KO epididymal sperm samples following two lysis protocols. In lysis protocol #1, sperm were re-suspended in 4X reducing SDS Sample Buffer …
Figure 1—figure supplement 3
Acrosomal cap localization of TMEM95 and IZUMO1 and IZUMO1 relocalization in WT and KO sperm.
(A) Immunocytochemistry images of WT sperm stained with PNA and IZUMO1 antibody (upper row) or PNA and TMEM95 antibody (lower row) in the absence of permeabilizing agents. Despite the absence of …
Figure 2 with 1 supplement
Reproductive performance of TMEM95-null male mice.
(A) Developmental outcomes following in vivo fertilization of WT eggs with WT, Hz or KO males. TMEM95-deficient males were unable to fertilize eggs, whereas no statistical differences were found in …
Figure 2—figure supplement 1
TMEM95 null sperm undergo acrosome reaction and bind to the egg membrane.
(A) Unfertilized eggs co-incubated with TMEM95 null sperm and stained with DAPI and PNA. All sperm that penetrated into the perivitelline space underwent the acrosome reaction (PNA negative) but …
Figure 3 with 2 supplements
TMEM95 does not interact with JUNO nor IZUMO1.
(A) Binding analysis using the AVEXIS assays shows that the soluble recombinant TMEM95 ectodomain does not interact with JUNO nor with IZUMO1. The entire ectodomains of the named proteins were …
Figure 3—figure supplement 1
Split GFP fusion assay.
(A) Amino acid sequence of FKBP1A underlined in blue fused via a short linker to the N-terminal region of GFP (GFP1-7) (green underline). (B) Amino acid sequence of the C-terminal region of GFP (GFP8…
Figure 3—figure supplement 2
Sequence alignments of TMEM95 and IZUMO1.
(A) Structure prediction of TMEM95 created by SWISS-MODEL web-based integrated service. Model TMEM95-P0DJF3 (left) was built using Template 5f4v.1.A (right) from Izumo1 sperm-egg fusion protein 1 (Oh…
Videos
Video 1
Confocal z-sections of an egg fixed in PFA following IVF with TMEM95-null sperm.
Multiple sperm are found in the perivitelline space, unable to fuse with the egg membrane.
Tables
Table 1
Details of peptides identified by MS/MS from 20 kDa gel band.
| Protein name | Score | Spi % | Sequence |
|---|---|---|---|
| TMEM95 | 5.33 | 68.8 | LLLCIFGIVLLsGVVSLQ |
| TMEM95 | 5.17 | 59.8 | LLLCIFGIVLLsGVVSLQ |
| TMEM95 | 4.12 | 46.1 | LLSGVVSLQVEY |
| TMEM95 | 3.42 | 53 | KTRYP |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Tmem95 KO | This article | Generated by CRISPR (M and M), available upn request | |
| Antibody | Rabbit Anti-TMEM95 | MyBioSource | MBS7004333 | |
| Antibody | Rabbit Anti-IZUMO1 | AbCam | Ab211623; RRID:AB_2650506 | |
| Antibody | Rat Anti-Izumo | Gift from Dr. Ikawa | Clone KS139-34 | |
| Antibody | Mouse Anti-Tubulin | Sigma | T8328, RRID:AB_1844090 | |
| Antibody | Rabbit anti-Flag | Sigma | F7425, RRID:AB_439687 | |
| Recombinant DNA reagent | Plasmid pMJ920 | (Jinek et al., 2013) | RRID:Addgene_42234 | |
| Recombinant DNA reagent | Plasmid px330 vector | (Yang et al., 2014) | RRID:Addgene_42230 | |
| Sequence-based reagent | Primers | This article | Detailed on M and M (ordered from Sigma) |
