(A) Bar graph displays the relative fluorescence unit (RFU) of endogenous full-length GIV protein in immunoblots of organ lysates published previously using three independent anti-GIV antibodies …
Quantitative immunoblotting of GIV in tissues.
Excel sheet with band densitometry values of immunoblots for endogenous GIV in various tissue lysates using three different anti-GIV/Girdin antibodies, as determined by ImageJ (corresponds to Figure 1A).
Full-length, uncropped immunoblots on mouse testis lysates with tGIV and pY GIV antibodies (corresponds to Figure 1G).
(A) The localization of G protein subunits and membrane and soluble adenylyl cyclases (mAC/sAC) and the calcium channel, Catsper, is shown. The intensity of the bar denotes the relative …
(A) CCDC88A expression profile was queried in the Human Protein Atlas. (B) GIV protein expression data is shown for each of the 44 tissues. (C) A summary of single-cell RNA (NX) from all single-cell …
(A) Schematic displays the approach used to search NCBI GEO database for testis and sperm transcriptomic datasets suitable to study correlations between the abundance of CCDC88A transcripts and male …
(A) Freshly ejaculated human sperm were segregated into low-motile and high-motile populations using ‘swim-up’ technique (see Materials and methods) and subsequently capacitated in vitro for 1 or 4 …
Full-length, uncropped immunoblots on human sperm lysates with tGIV and pY GIV antibodies (corresponds to Figure 3A).
Uncropped immunoblots on human sperm lysates with tGIV and pY GIV antibodies (corresponds to Figure 3B).
Full-length, uncropped immunoblots on sperm lysates with pan-pY and pY GIV antibodies (corresponds to Figure 3E).
(A) Schematic shows the domain map of GIV (top) and the evolutionarily conserved GEM motif within its C terminus. A functional GEM motif is required for GIV to bind and activate Gαi as well as bind …
(A) Schematic showing the workflow for fertility studies in conditional GIV-cKO mice. After intraperitoneal injection of tamoxifen, male mice were first primed in two phases—first by co-housing with …
Full-length, uncropped immunoblots on testes lysates with GIV and tubulin antibodies (corresponds to Figure 5D).
Excel sheet with time to live birth values observed in females co-housed with WT and GIV-cKO mice (corresponds to graphs in Figure 5E).
Excel sheet with litter size values from WT and GIV-cKO mice (corresponds to graphs in Figure 5F).
Excel sheet with sperm count values from WT and GIV-cKO mice (corresponds to graphs in Figure 5G).
Excel sheet with differential expression analysis-derived reactome pathway analyses of the most significantly up- and downregulated genes in GIV-cKO mice (corresponds to graphs in Figure 5I).
GIV was depleted in male mice by intraperitoneal injection of tamoxifen (see Figure 5A). Bar graphs show the testes weight (left; Figure 5—figure supplement 1—source data 1) and length (right; Figure…
Excel sheet with testes weight values from WT and GIV-cKO mice (corresponds to the left-side graph in Figure 5—figure supplement 1).
Excel sheet with testes length values from WT and GIV-cKO mice (corresponds to the left-side graph in Figure 5—figure supplement 1).
(A, B) Schematic (A, top) of cell-permeant His-TAT-GIV-CT wildtype (WT) and GEM-deficient mutant (F1685A; FA) peptides used in this work. Immunofluorescence images (B) representative of sperms after …
Full-length, uncropped immunoblots on GST pulldown assays (corresponds to Figure 6C).
Full-length, uncropped immunoblots on TAT-peptide-transduced sperm lysates with His, hexokinase, phospho-PKA substrate, phospho-Akt, and total Akt antibodies (corresponds to Figure 6D).
Full-length, uncropped immunoblots on TAT-peptide-transduced sperm lysates with pan-pY and pYGIV antibodies (corresponds to Figure 6E).
Excel sheet with sperm motility values (corresponds to graph in Figure 6G).
Excel sheet with sperm viability values (corresponds to graph in Figure 6H,I).
(A) Schematic summarizes the current knowledge of how Ca2+ and cAMP signaling regulates acrosome exocytosis during AR and how GIV’s ability to modulate cAMP via both Gαi/s is hypothesized to impact …
Excel sheet with cAMP concentrations (corresponds to graph in Figure 7B,C).
Excel sheet with % cells with various stages of acrosomal reaction (AR) (corresponds to graph in Figure 7D–F).
Excel sheet with % fertilized cells (corresponds to graph in Figure 7G–I).
(A) Bar graphs display the fold change in cAMP in mouse sperms in the presence (+) or absence (-) of various treatments (Figure 7—figure supplement 1—source data 1). (B) Acrosomal reaction was …
Excel sheet with cAMP concentrations (corresponds to graph in Figure 7—figure supplement 1).
Schematic summarizes the key findings in this work and places them in the context of existing literature. GIV is likely to primarily function during capacitation of sperm, during which it fulfills …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Antibody | Rabbit polyclonal anti-GIV (Girdin) (T-13) | Santa Cruz Biotechnology | sc-133371 | |
Antibody | Rabbit monoclonal diagnostic grade anti-Girdin/GIV antibody | Custom; Sprint Bioscience | SP173 | Validated in prior publication Ghosh et al., 2016 |
Antibody | Rabbit polyclonal anti-GIV (Girdin) (CC-Ab) | Millipore Sigma | ABT80 | |
Antibody | Rabbit polyclonal anti-GIV pS1675 Ab | Custom, from 21t Century Biosciences | n/a | Validated in prior publication Bhandari et al., 2015 |
Antibody | Rabbit polyclonal anti-GIV pS1689 Ab | Custom, from 21st Century Biosciences | n/a | Validated in prior publication López-Sánchez et al., 2013 |
Antibody | Rabbit monoclonal anti-GIV pY1764 Ab | Custom, Spring Biosciences Inc | n/a | Validated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016 |
Antibody | Rabbit monoclonal anti-pT308 AKT | Cell Signaling Technology | D9E | |
Antibody | Mouse monoclonal anti-total AKT | Cell Signaling Technology | 40D4 | |
Antibody | Mouse anti-sp56 | Thermo Fisher Scientific (Waltham, MA) | MA1-10866 | |
Antibody | Mouse anti- human hexokinase 1/2 monoclonal antibody | R&D Systems, (Minneapolis, MN) | MAB8179 | |
Antibody | Rabbit anti-phospho-PKA substrate (RRXS*/T*)100G7E | Cell Signaling Technology | 9624 | |
Antibody | Goat anti-rabbit IgG, Alexa Fluor 594 conjugated | ThermoFisher Scientific | A11072 | For immunofluorescence (IF) |
Antibody | Goat anti-mouse IgG, Alexa Fluor 488 conjugated | ThermoFisher Scientific | A11017 | For immunofluorescence (IF) |
Antibody | IRDye 800CW goat anti-mouse IgG secondary (1:10,000) | LI-COR Biosciences | 926-32210 | For immunoblotting |
Antibody | IRDye 680RD goat anti-rabbit IgG secondary (1:10,000) | LI-COR Biosciences | 926-68071 | For immunoblotting |
Strain, strain background (Mus musculus) | UbcCre-Ert2/+ xCcdc88afl/fl andCcdc88afl/fl mice | Masahide Takahashi (Nagoya University Graduate School of Medicine, Nagoya, Japan) | n/a | |
Strain, strain background (Mus musculus) | WT and GIV-cKO (conditional KO) mice | This work | UbcCre-Ert2/+ xCcdc88afl/fl (experimental group), and UbcCre-Ert2/ Cre-Ert2 x Ccdc88afl/fl (control group) | This work; male |
Strain, strain background (Mus musculus) | C57BL/6J mice (male and female) | The Jackson Laboratory | Stock number: 000664; Bar Harbor, ME | Male: source of sperm for biochemical, immunohistochemical, peptide transduction, and functional assaysFemale: for co-housing studies; source for eggs for IVF assays |
Chemical compound | Paraformaldehyde 16% | Electron Microscopy Biosciences | 15710 | |
Chemical compound | MTT | Millipore Sigma | 475989-1GM | |
Other | DAPI (4',6-Diamidino-2-Phenylindole, Dilactate) | Thermo Fisher Scientific | D3571 | Used in IF studies for staining DNA/nucleus |
Kit/reagent | HisPurä Cobalt Resin | Thermo Scientific | 89964 | |
Kit/reagent | Glutathione Sepharoseâ 4B | Sigma-Aldrich | GE17-0756-04 | |
Chemical compound | Protease inhibitor cocktail | Roche | 11873580001 | |
Chemical compound | Tyr phosphatase inhibitor cocktail | Sigma-Aldrich | P5726 | |
Chemical compound | Ser/Thr phosphatase inhibitor cocktail | Sigma-Aldrich | P0044 | |
Other | PVDF Transfer Membrane, 0.45 mM | Thermo Scientific | 88518 | Used for transfer in immunoblots |
Commercial assay or kit | Countess II Automated Cell Counter | Thermo Fisher Scientific | AMQAX1000 | |
Commercial assay or kit | Leica TCS SPE Confocal | Leica Microsystems | TCS SPE | |
Commercial assay or kit | Light Microscope (brightfield images) | Carl Zeiss LLC | Axio Observer, Inverted; 491917-0001-000 | |
Software | ImageJ | National Institute of Health | https://imagej.net/Welcome | |
Software | Prism | GraphPad | https://www.graphpad.com/scientific-software/prism/ | |
Software | LAS-X | Leica | https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls | |
Software | Illustrator | Adobe | https://www.adobe.com/products/illustrator.html | |
Software | ImageStudio Lite | LI-COR | https://www.licor.com/bio/image-studio-lite/ |