ECB-ART-47526BMC Biol January 1, 2019; 17 (1): 86.
GLIPR1L1 is an IZUMO-binding protein required for optimal fertilization in the mouse.
BACKGROUND: The sperm protein IZUMO1 (Izumo sperm-egg fusion 1) and its recently identified binding partner on the oolemma, IZUMO1R, are among the first ligand-receptor pairs shown to be essential for gamete recognition and adhesion. However, the IZUMO1-IZUMO1R interaction does not appear to be directly responsible for promoting the fusion of the gamete membranes, suggesting that this critical phase of the fertilization cascade requires the concerted action of alternative fusogenic machinery. It has therefore been proposed that IZUMO1 may play a secondary role in the organization and/or stabilization of higher-order heteromeric complexes in spermatozoa that are required for membrane fusion. RESULTS: Here, we show that fertilization-competent (acrosome reacted) mouse spermatozoa harbor several high molecular weight protein complexes, a subset of which are readily able to adhere to solubilized oolemmal proteins. At least two of these complexes contain IZUMO1 in partnership with GLI pathogenesis-related 1 like 1 (GLIPR1L1). This interaction is associated with lipid rafts and is dynamically remodeled upon the induction of acrosomal exocytosis in preparation for sperm adhesion to the oolemma. Accordingly, the selective ablation of GLIPR1L1 leads to compromised sperm function characterized by a reduced ability to undergo the acrosome reaction and a failure of IZUMO1 redistribution. CONCLUSIONS: Collectively, this study characterizes multimeric protein complexes on the sperm surface and identifies GLIPRL1L1 as a physiologically relevant regulator of IZUMO1 function and the fertilization process.
PubMed ID: 31672133
PMC ID: PMC6824042
Article link: BMC Biol
Genes referenced: aqr LOC100888399 LOC115919139 LOC588678 LOC593642 skiv2l
Article Images: [+] show captions
|Fig. 1. Identification of mouse sperm multimeric protein complexes with affinity for homologous oolemmal proteins. a Mouse spermatozoa were purified under non-capacitating (Non-Cap) or capacitating (Cap) conditions. A portion of the latter population was also challenged with A23187 to induce the acrosome reaction (AR). To detect native protein complexes with affinity for oolemmal proteins, far-western blotting with biotin-labeled preparations of oocyte lysates (Far-Western) was undertaken. Four predominant oolemmal protein-binding complexes (arrowheads, I–IV) were identified. Each experiment was replicated a minimum of three times and representative gels and blots are shown. The numbers on the left correspond to the molecular weight (kDa) of native PAGE protein standards. b Validation of GLIPR1L1 and IZUMO1 antibodies. The specificity of the antibodies used in this study was confirmed by immunoblotting against sperm protein extracts. This experiment was replicated three times and immunoblots are shown. The numbers on the left correspond to the molecular weight of the protein standards. c Identification of mouse sperm protein complexes comprising IZUMO1 and GLIPR1L1. Populations of non-capacitated (Non-Cap), capacitated (Cap), and acrosome-reacted (AR) mouse spermatozoa were solubilized in blue native lysis buffer. The extracted proteins were resolved on BN-PAGE gels before being prepared for immunoblotting with either IZUMO1 or GLIPR1L1 antibodies. Arrowheads indicate the predominant complexes recognized by each antibody. Red arrowheads correspond to complexes (I and IV) that co-migrated with those that bound oolemmal proteins (see Fig. 1a). Each experiment was replicated a minimum of three times and representative images are shown. The numbers on the left correspond to the molecular weight (kDa) of native PAGE protein standards|
|Fig. 2. IZUMO1 and GLIPR1L1 reside in multimeric protein complexes. a Native protein complexes were extracted from acrosome-reacted mouse spermatozoa and resolved by BN-PAGE. b A single lane of the BN-PAGE gel was then placed atop an SDS-PAGE gel and the individual proteins within each complex resolved according to their molecular weight. c Gels were then used for immunoblotting with IZUMO1 or GLIPR1L1 antibodies. Each of these experiments was repeated three times and representative images are shown. The boxed section indicates the position of labeled proteins vertically aligned with complexes I and IV|
|Fig. 3. Interaction between IZUMO1 and GLIPR1L1 using reciprocal co-immunoprecipitation (IP). Acrosome-reacted mouse spermatozoa were subjected to immunoprecipitation using IZUMO1 or GLIPR1L1 antibodies as described in the “Methods” section. Membranes were probed with the target antibody, to confirm the efficacy of immunoprecipitation, before being stripped and re-probed with the alterative antibody to confirm the target protein interaction. Whole sperm lysate was included to confirm the identity of the co-precipitated proteins, as was the material recovered after washing the beads to confirm the specificity of the elution. This experiment was replicated three times and representative blots are depicted|
|Fig. 4. IZUMO1 and GLIPR1L1 localization in developing mouse germ cells. a Enriched populations of pachytene spermatocytes and b round spermatids were purified and sequentially labeled with IZUMO1 or GLIPR1L1 and appropriate Alexa Fluor 488-conjugated secondary antibodies (green) followed by Alexa Fluor 594-conjugated PNA (red). Labeling was also conducted on capacitated spermatozoa that were either c acrosome intact or d acrosome reacted. This experiment was replicated three times, and representative images are shown. Scale bar = 10 μm|
|Fig. 6. IZUMO1 and GLIPR1L1 are present within the membrane raft in live capacitated spermatozoa. The presence of IZUMO1 and GLIPR1L1 was confirmed by colocalization with the raft marker, GM1. Membrane rafts were visualized in live cells by staining with Alexa Fluor 555-labeled cholera toxin B subunit (red). The cells were then fixed and labeled with the appropriate primary and Alexa Fluor 488-conjugated secondary antibodies (green). This experiment was replicated three times with a minimum of 200 spermatozoa being examined in each. Representative images are shown. Scale bar = 10 μm|
References [+] :
Aguilar PS, Genetic basis of cell-cell fusion mechanisms. 2013, Pubmed