Gaikwad AS , Anderson AL , Merriner DJ , O'Connor AE , Houston BJ , Aitken RJ , O'Bryan MK , Nixon B .

	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. 5. Co-localization of IZUMO1 and GLIPR1L1 in developing mouse germ cells using an in situ proximity ligation assay. a The cells were counterstained with DAPI (blue) and PNA (green). This experiment was replicated three times, and representative images are shown. b In the case of capacitated spermatozoa, a lower magnification image is also included to highlight the differences in PLA labeling between acrosome intact and acrosome-reacted (arrowhead) spermatozoa. Scale bar = 10 μm. c The percentage of cells displaying PLA positive labeling was recorded. Each experiment was replicated three times and the data are expressed as the mean ± S.E.M. *P < 0.05, compared with spermatocytes. Individual data points for each replicate are provided in Additional file 6: Raw data
	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
	Fig. 7. Glipr1l1 expression and immunofluorescent localization in mouse sperm from wild type and Glipr1l1−/− mice. a qPCR analysis of Glipr1l1 mRNA levels in isolated testis and germ cells from Glipr1l1−/− mice relative to wild type (WT) mice. mRNA expression levels were normalized to the housekeeping gene Ppia. This experiment was replicated three times and data is shown as mean ± S.D., **** P < 0.0001. Individual data points for each replicate are provided in Additional file 6: Raw data. b Localization of GLIPR1L1 at the sperm head and at the connecting piece. GLIPR1L1 staining (red, marked with white arrows) was observed in WT sperm and no staining was observed in the Glipr1l1−/− sperm. In all the images, nuclear DNA was stained with DAPI (blue). This experiment was replicated three times and representative images are shown. Scale bar = 20 μm
	Fig. 8. Fertility assessment of Glipr1l1−/− mice. a Testicular morphology was assessed by periodic acid-Schiff (PAS) staining of the testis sections from WT and Glipr1l1−/− mice as described in the “Methods” section. This experiment was replicated in a minimum of three mice per genotype and representative PAS staining is shown. Data are expressed as the mean ± S.D. Scale bar = 50 μm. b Daily sperm production (DSP) within testis from WT and Glipr1l1−/− mice. This experiment was replicated in a minimum of five mice per genotype, and the data are expressed as the mean ± S.D. Genotypes are shown on the X-axis, and data were shown as mean ± S.D. c–e Computer-assisted sperm analysis demonstrates no significant difference in the motility, progressively motility, or other sperm velocity parameters between WT and Glipr1l1−/− mice. This experiment was replicated in a minimum of five mice per genotype, and the data are expressed as the mean ± S.D. f As a marker of capacitation, the level of global tyrosine phosphorylation in the whole sperm population was measured. The X-axis depicts the length of time sperm were exposed to capacitation permissive media, as described in the “Methods” section. The relative intensity of the 70-kDa (p70) band was measured. This experiment was replicated in a minimum of six mice per genotype and the data are expressed as the mean ± S.D. g, h Assessment of the ability of Glipr1l1−/− sperm to undergo the acrosome reaction. AR indicates acrosome-reacted spermatozoa in which the entire acrosomal contents had been released from the sperm head (i.e., no PNA labeling). Partial AR indicates spermatozoa in which the acrosomal contents were incompletely shed from the sperm head (i.e., partial PNA labeling). No AR indicates spermatozoa in which the acrosomal contents are retained (i.e., complete PNA labeling). + or – indicates exposure to progesterone for the final 15 min of capacitation. This experiment was replicated in a minimum of four mice per genotype with a minimum of 200 spermatozoa being examined in each replicate. Data are expressed as the mean ± S.D. *P < 0.05, **P < 0.01, ***P < 0.001. Individual data points for each replicate are provided in Additional file 6: Raw data
	Fig. 9. Reduced fertilization potential of male Glipr1l1−/− mice. a The absence of GLIPR1L1 did not significantly affect the number of sperm that could bind to the zona pellucida in an IVF assay. b The percentage of two-cell embryos observed following IVF using sperm from Glipr1l1−/− mice compared to sperm from WT controls. Both experiments were replicated in five mice per genotype and the data are expressed as the mean ± S.D. *P < 0.05. Individual data points for each replicate are provided in Additional file 6: Raw data
	Fig. 10. GLIPR1L1 loss disrupts IZUMO1 redistribution during the acrosome reaction. a Sperm were stained for IZUMO1 localization and the acrosome was labeled with PNA (green), while DNA was stained with DAPI (blue). Scale bar = 20 μm. b The percentage of sperm that displayed IZUMO1 relocalization was scored in non-acrosome-reacted (Non-AR) and acrosome-reacted (AR) sperm from WT and Glipr1l1−/− mice. This experiment was replicated five times with a minimum of 200 spermatozoa being examined in each. Representative images are shown. *P < 0.05, **P < 0.01, ***P < 0.001. Individual data points for each replicate are provided in Additional file 6: Raw data

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