Herr Identifying and Screening

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Presented by Dr. John Herr at the Alliance for Contraception in Cats & Dogs’ 4th International Symposium on Non-Surgical Contraceptive Methods of Pet Population Control, April 8-10, 2010, in Dallas, Texas, U.S.

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Slide 1: 

Alliance for Contraception in Cats and Dogs April 10, 2010 John C. Herr, Ph.D. Center for Research in Contraceptive and Reproductive Health Department of Cell Biology University of Virginia Health System Identifying and Screening Targets for Contraceptive Impact Model Molecule: The Oocyte Specific Drugable Contraceptive Target SAS1R

Slide 2: 

Sperm–oolemma interactions Adapted from: R. Yanagimachi, Sperm-egg fusion. In Current topics in membranes and transport, Academic Press, San Diego, CA (1988).

SAS1R Sperm Acrosomal SLLP1 Receptor : 

SAS1R Sperm Acrosomal SLLP1 Receptor Oocyte specific membrane receptor [binding protein] for sperm ligand SLLP1. SAS1R appears first during oogenesis in secondary follicles. Oolemmal metalloprotease functioning in sperm-oocyte binding.

Mouse SLLP1 in Sperm Acrosome : 

Mouse SLLP1 in Sperm Acrosome SLLP1 was localized mainly to the anterior acrosome (arrowheads), with some sperm showing staining in the equatorial segment (arrow).

SLLP1 is Retained in Sperm Tightly Bound to the Oolemma : 

SLLP1 is Retained in Sperm Tightly Bound to the Oolemma When imaged in their respective focal planes all sperm tightly bound to the egg displayed mSLLP1 staining. The insert in panel (D) is an enlarged view of one sperm bound to the oolemma (indicated with arrow in panel D). SYTOX ANTI-SLLP1

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In acrosome reacted sperm SLLP1 is retained in the equatorial segment- the region that fuses with the oolemma Recombinant SLLP1 and anti-SLLP1 block in vitro fertilization Dose dependent inhibition of sperm binding and fusion by rec mouse SLLP1

SLLP1 Binding Sites on Mouse Eggs : 

SLLP1 Binding Sites on Mouse Eggs Indirect immunofluorescence of mSLLP1 complementary binding sites on the egg surface. (A) Mature unfertilized or (B) fertilized mouse eggs, with (i) or without (ii) zona pellucida, were incubated with 100 μg/ml recmSLLP1 for 45 min, washed, and exposed to anti-recmSLLP1 sera followed by secondary. Mouse SLLP1 binding sites were localized in the perivitelline space and on the microvillar region of unfertilized oocytes. In fertilized eggs, staining was observed along the entire plasma membrane of both zona-free and zona intact oocytes. Hypotheses: SLLP1 receptors on microvillar domain. SLLP1 receptors redistribute after fertilization.

SAS1R Splice Variants, Mouse : 

SAS1R Splice Variants, Mouse Six SAS1R variants cloned, 3 with signal sequences: Variants 3 and 4 showed 34 aa deletions from exons 4 and 5. Variants 5 and 6 showed 31 aa deletions and 9 aa insertion in 5th exon. Proform Sizes: V1: 435—412aa [G-A]; V2: 414, V3: 380: V4: 402-381 [G-A]; V5: 392; V6:413-390 [G-A]. SAS1R has putative transmembrane domain, shaded. SAS1R is a metalloprotease in the MA clan, family M12A, astacin. The Zn metal ion binding catalytic pocket is shown in underline. The consensus motif HEXXHXXGXXH with histidine residues for Zn coordination and conserved catalytic residue, E [glutamic acid] forms part of the catalytic pocket along with a tyrosine zinc ligand embedded in the motif [SXHHY].  1 23 120 143/163 182 240 435

SAS1R Purification and Antibody Production : 

SAS1R Purification and Antibody Production A: Purification of SAS1R by affinity chromatography, Coomassie stain of uninduced (U), induced (I) and purified (P) fraction from E. coli extract. B: Anti-his Western analysis of induced and purified SAS1R. C: Western analysis of recombinant SAS1R using guinea pig preimmune (PI) and immune (IM) sera. D: Western analysis of 150 zona intact (ZIE) and 150 zona free (ZFE) mouse eggs with PI and IM sera.

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Brain (whole) Stomach (whole) Intestine (whole) Colon Liver Lung Kidney Heart Ovary Skeletal muscle Spleen Testis Thymus Uterus (non-pregnant) Placenta (late pregnancy) mSAS1R Actins mSAS1R Multi Tissue Northern Blot

Stages of mouse folliculogenesis : 

Stages of mouse folliculogenesis

Day 0 : 

Day 0 GP mSAS1R PI 1:500 GP mSAS1R IM 1:500

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GP mSAS1R PI 1:500 GP mSAS1R IM 1:500 Day 1.5

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Anti GP pAb to mSAS1R Pre-immune GP mSAS1R IM 1:500 Day 2

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Anti GP pAb to mSAS1R Pre-immune GP mSAS1R IM 1:500 Day 3

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GP mSAS1R PI 1:500 GP mSAS1R IM 1:500 Day 4

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GP mSAS1R PI 1:500 GP mSAS1R IM 1:500 Day 7

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GP mSAS1R PI 1:500 GP mSAS1R IM 1:500 Day 14

Day 28 : 

GP mSAS1R PI 1:500 GP mSAS1R IM 1:500 Day 28

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GP mSAS1R PI 1:500 GP mSAS1R IM 1:500 Day 56

Cat SAS1R is a ~45 kDa protein : 

Cat SAS1R is a ~45 kDa protein

Cat SAS1R immunolocalization : 

Pre-Immune Guinea Pig anti-mSAS1R sera at 1:200 dilution does not immunostains the cat ooplasm of any ovarian follicles indicated in black arrows. X200 magnification. Immune Guinea Pig anti-mSAS1R sera at 1:200 dilution immunostains the cat ooplasm of secondary and tertiary follicles indicated in red arrows. X200 magnification. Primordial and primary oocytes do not express this protein. Cat SAS1R immunolocalization

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Immune Guinea Pig anti-mSAS1R sera at 1:200 dilution immunostains the dog ooplasm of secondary and tertiary follicles indicated in red arrows. X200 magnification. Primordial and primary oocytes do not express this protein. Pre-Immune Guinea Pig anti-mSAS1R sera at 1:200 dilution does not immunostains the dog ooplasm of any ovarian follicles indicated in black arrows. X200 magnification. Dog SAS1R immunolocalization

SAS1R Localization on Live Oocytes: Microvillar Domain of Oolemma : 

SAS1R Localization on Live Oocytes: Microvillar Domain of Oolemma Panel B: In un-permeablized zona intact ovulated oocytes arrested in M2 (pre- fertilization) stage SAS1R IF (IM) concentrated in a dome shaped microvillar domain on the surface of the oocyte plasma membrane. Blue colored regions stained with DAPI correspond to eccentrically positioned nucleus arrested in M2 antipodal to the SAS1R positive domain. Control oocytes stained with pre-immune serum (PI) showed no immunoreactivity.

SAS1R Distribution: Confocal Imaging Before and After Fertilization : 

SAS1R Distribution: Confocal Imaging Before and After Fertilization Undergoes transition from cytoplasm in GV oocytes to oolemma in M2. SAS1R is concentrated on oolemma of ovulated oocyte—appears to function at time of fertilization.

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SAS1R Model: Zn binding active site cleft is formed by two distinct N-terminal and C-terminal domains on either side and lined by evolutionarily conserved histidine residues [right: green]. Of four highly conserved histidines highlighted three [H161, H165 and H171] are predicted with high confidence to be involved in Zn coordination [the blue green ball].

Recombinant SAS1R is Proteolytically Active : 

Recombinant SAS1R is Proteolytically Active Protease activities of purified recombinant mouse SAS1R or SLLP1 utilizing a Fluorescent tagged synthetic peptide substrate using FRET assay. (A) Enzymatic activity of mouse recombinant SAS1R after one hour. (B) Enzymatic activities of varying concentrations of mouse recombinant SAS1R and recombinant SLLP1 after 24 hours. A concentration dependent cleavage of synthetic peptide was observed with SAS1R but not with SLLP1 at the 24 hr time point. Proteolytic activity of SAS1R was observed within the first hour and increased over the period of time and almost saturated by 24 hours.

Evidence SAS1R is Receptor for SLLP1 : 

Evidence SAS1R is Receptor for SLLP1 Native SAS1R showed binding to rSLLP1 by surface plasmon resonance technique. Bound rec SAS1R captured rec SLLP1 in membrane overlay assay (Far Western analysis). SAS1R and SLLP1 revealed molecular binding properties by yeast two hybrid analysis. Immunoprecipitation of recSAS1R recovered rec SLLP1 and immunoprecipitated rec SLLP1 recovered rec SAS1R from rabbit reticulocyte extract. recSLLP1 binds to oocyte microvillar domain and co-localizes with native SAS1R. recSAS1R binds to acrosome and co-localizes with native SLLP1. Native SLLP1 from sperm acrosomal matrix localizes with native SAS1R. Native SAS1R and native SLLP1 are co-precipitated from mixtures of non-ionic detergent extracts of oocytes and sperm.

Summary : 

Summary SAS1R Active metalloproteinase with unique N- and C- terminal regions. M12A family, clan MA Conserved across phyla being found in mammals and invertebrates. Mammalian forms have putative TM domain apparently lacking in lower organisms. In mice SAS1R has 6 alternative splice variants. Ovary and Oocyte specific protein. Expression initiated in bilaminar secondary follicles. SAS1R persists in oocytes through ovulation and fertilization

Summary : 

Summary SAS1R Found on the surface of transfected CHOK1 cells. Transmembrane domain functional or bound to some other carrier. Undergoes change in intercellular localization with M1—M2 transition. Becomes localized on the microvillar domain of oolemma in mature oocytes (MII). Levels go down following fertilization and the protein is absent in blastocysts. recSAS1R treatment of sperm blocks in IVF.

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Properties of an Ideal Contraceptive Target Molecule or Pathway Unique to the Cell Type Targeted Opportunity for Selective Targeting While Minimizing Side-effects on Non-reproductive Tissues Drugable or Targetable Contains functional domains amenable to blocking or binding; including accessibility at the target cell surface Reversibility [key for Contraception not Sterilization] Properties of the target allow for restoration of fertility.

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Hypothalamus Pituitary Ovary P- Estrogens Testosterone Progestins . Disadvantages of Steroids: Act on many organ systems including brain, uterus, breast, muscle, liver, skin. Concerns over increased risk of female reproductive cancers.

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SAS1R Homology Model Future Work: Small Molecule Inhibitors of SAS1R

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Paradigm Shift: Contraceptive Drug that Acts Directly on the Oocyte. Depot or oral non-steroidal, small molecule contraceptive antagonist that acts directly on the ovary to inhibit oocyte maturation, ovulation and fertilization.

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