GIV/Girdin, a non-receptor modulator for Gαi/s, regulates spatiotemporal signaling during sperm capacitation and is required for male fertility

  1. Sequoyah Reynoso
  2. Vanessa Castillo
  3. Gajanan Dattatray Katkar
  4. Inmaculada Lopez-Sanchez
  5. Sahar Taheri
  6. Celia Espinoza
  7. Cristina Rohena
  8. Debashis Sahoo
  9. Pascal Gagneux  Is a corresponding author
  10. Pradipta Ghosh  Is a corresponding author
  1. Department of Pathology, School of Medicine, University of California San Diego, United States
  2. Department of Cellular and Molecular Medicine, School of Medicine, University of California San Diego, United States
  3. Department of Medicine, School of Medicine, University of California San Diego, United States
  4. Department of Computer Science and Engineering, Jacob’s School of Engineering, University of California San Diego, United States
  5. Moore’s Comprehensive Cancer Center, University of California San Diego, United States
  6. Department of Pediatrics, School of Medicine, University of California San Diego, United States
  7. Veterans Affairs Medical Center, United States
8 figures, 1 table and 1 additional file

Figures

Figure 1 with 2 supplements
GIV (CCDC88A) is highly expressed in spermatocytes in testis and localizes to the acrosomal cap.

(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 …

Figure 1—source data 1

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).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig1-data1-v1.xlsx
Figure 1—source data 2

Full-length, uncropped immunoblots on mouse testis lysates with tGIV and pY GIV antibodies (corresponds to Figure 1G).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig1-data2-v1.pptx
Figure 1—figure supplement 1
Schematic summarizing the known localization (A) and role of G proteins/AC proteins and their impact on cAMP signaling (B) during sperm processes.

(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 …

Figure 1—figure supplement 2
CCDC88A (GIV/Girdin) is highly expressed in the testes, most specifically the spermatocytes.

(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 …

Transcripts of CCDC88A (GIV) are downregulated in infertile male testis and semen.

(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 …

GIV localizes to the head and tail of human and murine sperms and is rapidly tyrosine phosphorylated during capacitation.

(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 …

Figure 3—source data 1

Full-length, uncropped immunoblots on human sperm lysates with tGIV and pY GIV antibodies (corresponds to Figure 3A).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig3-data1-v1.pptx
Figure 3—source data 2

Uncropped immunoblots on human sperm lysates with tGIV and pY GIV antibodies (corresponds to Figure 3B).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig3-data2-v1.pptx
Figure 3—source data 3

Full-length, uncropped immunoblots on sperm lysates with pan-pY and pY GIV antibodies (corresponds to Figure 3E).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig3-data3-v1.pptx
GIV’s guanine nucleotide-exchange modulator (GEM) function is dynamically phosphoregulated during capacitation and acrosomal reaction (AR) in a spatiotemporally segregated manner.

(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 …

Figure 5 with 1 supplement
GIV is required for fertility in male mice.

(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 …

Figure 5—source data 1

Full-length, uncropped immunoblots on testes lysates with GIV and tubulin antibodies (corresponds to Figure 5D).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig5-data1-v1.pptx
Figure 5—source data 2

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).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig5-data2-v1.xlsx
Figure 5—source data 3

Excel sheet with litter size values from WT and GIV-cKO mice (corresponds to graphs in Figure 5F).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig5-data3-v1.xlsx
Figure 5—source data 4

Excel sheet with sperm count values from WT and GIV-cKO mice (corresponds to graphs in Figure 5G).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig5-data4-v1.xlsx
Figure 5—source data 5

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).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig5-data5-v1.xlsx
Figure 5—figure supplement 1
GIV is required for male fertility, but its depletion does not impact testes weight or length.

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…

Figure 5—figure supplement 1—source data 1

Excel sheet with testes weight values from WT and GIV-cKO mice (corresponds to the left-side graph in Figure 5—figure supplement 1).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig5-figsupp1-data1-v1.xlsx
Figure 5—figure supplement 1—source data 2

Excel sheet with testes length values from WT and GIV-cKO mice (corresponds to the left-side graph in Figure 5—figure supplement 1).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig5-figsupp1-data2-v1.xlsx
GIV’s GEM function is required for sperm motility and survival during capacitation.

(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 …

Figure 6—source data 1

Full-length, uncropped immunoblots on GST pulldown assays (corresponds to Figure 6C).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig6-data1-v1.pptx
Figure 6—source data 2

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).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig6-data2-v1.pptx
Figure 6—source data 3

Full-length, uncropped immunoblots on TAT-peptide-transduced sperm lysates with pan-pY and pYGIV antibodies (corresponds to Figure 6E).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig6-data3-v1.pptx
Figure 6—source data 4

Excel sheet with sperm motility values (corresponds to graph in Figure 6G).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig6-data4-v1.xlsx
Figure 6—source data 5

Excel sheet with sperm viability values (corresponds to graph in Figure 6H,I).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig6-data5-v1.xlsx
Figure 7 with 1 supplement
GIV’s GEM function inhibits acrosomal reaction (AR).

(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 …

Figure 7—source data 1

Excel sheet with cAMP concentrations (corresponds to graph in Figure 7B,C).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig7-data1-v1.xlsx
Figure 7—source data 2

Excel sheet with % cells with various stages of acrosomal reaction (AR) (corresponds to graph in Figure 7D–F).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig7-data2-v1.xlsx
Figure 7—source data 3

Excel sheet with % fertilized cells (corresponds to graph in Figure 7G–I).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig7-data3-v1.xlsx
Figure 7—figure supplement 1
An intact GEM motif in GIV is required for inhibiting cAMP surge and acrosomal reaction.

(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 …

Figure 7—figure supplement 1—source data 1

Excel sheet with cAMP concentrations (corresponds to graph in Figure 7—figure supplement 1).

https://cdn.elifesciences.org/articles/69160/elife-69160-fig7-figsupp1-data1-v1.xlsx
Summary and working model: spatiotemporally segregated roles of GIV/Girdin during sperm capacitation.

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 …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
AntibodyRabbit polyclonal anti-GIV (Girdin) (T-13)Santa Cruz Biotechnologysc-133371
AntibodyRabbit monoclonal diagnostic grade anti-Girdin/GIV antibodyCustom; Sprint BioscienceSP173Validated in prior publication Ghosh et al., 2016
AntibodyRabbit polyclonal anti-GIV (Girdin) (CC-Ab)Millipore SigmaABT80
AntibodyRabbit polyclonal anti-GIV pS1675 AbCustom, from 21t Century Biosciencesn/aValidated in prior publication Bhandari et al., 2015
AntibodyRabbit polyclonal anti-GIV pS1689 AbCustom, from 21st Century Biosciencesn/aValidated in prior publication López-Sánchez et al., 2013
AntibodyRabbit monoclonal anti-GIV pY1764 AbCustom, Spring Biosciences Incn/aValidated in prior publications Midde et al., 2015; Lin et al., 2011; Midde et al., 2018; Dunkel et al., 2016
AntibodyRabbit monoclonal anti-pT308 AKTCell Signaling TechnologyD9E
AntibodyMouse monoclonal anti-total AKTCell Signaling Technology40D4
AntibodyMouse anti-sp56Thermo Fisher Scientific (Waltham, MA)MA1-10866
AntibodyMouse anti- human hexokinase 1/2 monoclonal antibodyR&D Systems, (Minneapolis, MN)MAB8179
AntibodyRabbit anti-phospho-PKA substrate (RRXS*/T*)100G7ECell Signaling Technology9624
AntibodyGoat anti-rabbit IgG, Alexa Fluor 594 conjugatedThermoFisher ScientificA11072For immunofluorescence (IF)
AntibodyGoat anti-mouse IgG, Alexa Fluor 488 conjugatedThermoFisher ScientificA11017For immunofluorescence (IF)
AntibodyIRDye 800CW goat anti-mouse IgG secondary (1:10,000)LI-COR Biosciences926-32210For immunoblotting
AntibodyIRDye 680RD goat anti-rabbit IgG secondary (1:10,000)LI-COR Biosciences926-68071For immunoblotting
Strain, strain background (Mus musculus)UbcCre-Ert2/+ xCcdc88afl/fl andCcdc88afl/fl miceMasahide Takahashi (Nagoya University Graduate School of Medicine, Nagoya, Japan)n/a
Strain, strain background (Mus musculus)WT and GIV-cKO (conditional KO) miceThis workUbcCre-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 LaboratoryStock number: 000664; Bar Harbor, MEMale: source of sperm for biochemical, immunohistochemical, peptide transduction, and functional assaysFemale: for co-housing studies; source for eggs for IVF assays
Chemical compoundParaformaldehyde 16%Electron Microscopy Biosciences15710
Chemical compoundMTTMillipore Sigma475989-1GM
OtherDAPI (4',6-Diamidino-2-Phenylindole, Dilactate)Thermo Fisher ScientificD3571Used in IF studies for staining DNA/nucleus
Kit/reagentHisPurä Cobalt ResinThermo Scientific89964
Kit/reagentGlutathione Sepharoseâ 4BSigma-AldrichGE17-0756-04
Chemical compoundProtease inhibitor cocktailRoche11873580001
Chemical compoundTyr phosphatase inhibitor cocktailSigma-AldrichP5726
Chemical compoundSer/Thr phosphatase inhibitor cocktailSigma-AldrichP0044
OtherPVDF Transfer Membrane, 0.45 mMThermo Scientific88518Used for transfer in immunoblots
Commercial assay or kitCountess II Automated Cell CounterThermo Fisher ScientificAMQAX1000
Commercial assay or kitLeica TCS SPE ConfocalLeica MicrosystemsTCS SPE
Commercial assay or kitLight Microscope (brightfield images)Carl Zeiss LLCAxio Observer, Inverted; 491917-0001-000
SoftwareImageJNational Institute of Healthhttps://imagej.net/Welcome
SoftwarePrismGraphPadhttps://www.graphpad.com/scientific-software/prism/
SoftwareLAS-XLeicahttps://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls
SoftwareIllustratorAdobehttps://www.adobe.com/products/illustrator.html
SoftwareImageStudio LiteLI-CORhttps://www.licor.com/bio/image-studio-lite/

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