Fertilization

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Fertilization

• Sperm/Egg
• recognition
• acrosomal
• reaction

• Gamete fusion
• prevention
• of polyspermy

• Egg activation
• activation of
• preformed mRNA

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Egg recognition proteins (Bindin)
Cumulus zona lysins
Cumulus cells
Perivitelline space
Egg plasma membrane
Zona pellucida
Ovum
Spermatozoan
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Antiserum
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Serum Antibody to Rabbit Gamma Globulin (Ab2)
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Diaminobenzidine
peroxidase
Antigen bindin
Brown precipitate
2nd Ab Pig anti-rabbit Ab
1st Ab Rabbit anti-bindin Ab
Fluorsceine Dye
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Mouse embryonic stem cells stained with a
fluorescent green marker for embryonic germ
cells (precursor sex cells).
Mouse melanoma M3 double labeling with j-Red and
phi-Yellow-tagged mitochondria
Nuclear components
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Biotin
Avidin 4 subunits with high affinity for
biotin
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Monoclonal Antibodies
Myeloma cells unable to make their own
Abs -immortal due to presence of
viral oncogenes
Normal B-lymphocytes - short lifespan
(hypoxanthine/aminopterin/ thymidine containing
medium)
de novo synthesis of purines
and pyrimidines
Purines Pyrimidines
HPRT Salvage enzyme
X
DNA synthesis
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Perivitelline space
Meiosis II
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Sperm/Egg Recognition
Oocyte
Galatosyltransferase catalyzes addition
of galactose to carbohydrate chain terminating
in N-acetylglucosamine -no free galactose
therefore enzyme will bind to
N-acetylglucosamine but not release
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Male Contraception
Adjudin

• Adjudin disrupts cell connections
• between Sertoli cells developing
• sperm cells thereby disrupting support
• - Body clears this molecule incredibly
• rapidly thus delivery is a problem

Sertoli (support) cells

• Attach adjudin to a modified FSH
• molecule FSH does not retain its
• biological activity but is still recognized
• by the receptors on the Sertoli cells
• Perfect delivery system
• - Reversible!!!

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Sea Urchin
DAB H2O2
DAB Diaminobenzidine H2O2 Hydrogen peroxide
- Tagged double antibody localization of antigen
-
Fig. 7.13, pg. 185
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Sea Urchin
Mouse
Fig. 7.8, pg. 182
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Acrosomal reaction in the sea urchin
Actin filament
Rising pH -------- dissociation of
profilin from actin
Binding site for protein on actin molecule
Profilin 11
Fig. 7.11, pg. 184
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Activation of dynein ATPase Dynein protein bound
to alpha-tubulin alpha-tubulin and beta-tubulin
coupled together
Microtubules
Fig. 7.3, pg. 178
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Sperm
Oolemma
Zona pellucida
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Signal Transduction
(protease)
G proteins complex with receptors and form a
GTP- bound molecule
Inositol triphosphate binds to ER receptors and
opens Ca channels
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PLC phospholipase PIP2 phosphatidylinositol-
4,5 biphosphate DAG
diacylglycerol IP3 Inositol triphosphate
Fig. 7.26, pg. 197
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PLC phospholipase C PIP2 phosphatidylinositol-
4,5 biphosphate DAG
diacylglycerol IP3 Inositol triphosphate PKC
Protein kinase C
Fast Block to Polyspermy
PKC Protein kinase C
PKC

pH
allosteric regulator
Fig. 7.24, pg. 196
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Na


-70 mV to 20 mV
Linearization of intermediate filaments/formation
of actin microfilaments
Fig. 7.22, pg. 194
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Study of fertilization-induced Ca flux
Time-lapse confocal microscopy Eggs
injected with Ca-sensitive fluorochrome calcium
green dextran Scan cell at 5? thicknesses
and produce 3-D image
(similar to Fig. 7.20, pg. 192)
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Redistribution of PKC following Ca release
Single 0.5 ? section
Stack of 10 0.5? sections of unactivated egg
diffuse distribution of PKC
Stack of 10 0.5? sections from an activated egg
PKC localized along plasma membrane

• A PKC inhibitor which binds at the catalytic
  site of PKC and
• fluoresces used as a probe
• Protein kinase C phosphorylating substances at
  membrane surface
• --- PKM (cytosolic counterpart) remodels
  internal cytoskeleton

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Fast Block to Polyspermy
PKC Protein kinase C
PKC
Fig. 7.24, pg. 196
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Perivitelline space
Bindin receptors
PKC induces exocytosis of cortical granules and
polymerization of actin filaments which induces
formation of 2nd polar body
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NADP is coenzyme for lipid biosynthesis
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1st Polar body
Male pronucleus
Female pronucleus
2nd Polar body
Male pronucleus
Female pronucleus
ZP
Male DNA tightly packaged by protamines Glutathi
one in egg reduces disulfide bonds and
uncoils this DNA reestablishes histone
proteins
Chromosomes orient on a common mitotic spindle
for first cell division
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Cycloheximide
Leucine incorporation, fertilized eggs
Leucine incorporation, unfertilized eggs
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Sea urchin eggs
Fig. 7.23, pg. 194
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Zona pellucida
Blastula
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Sperm/Egg Recognition
Species Specificity
Oocyte
Galatosyltransferase catalyzes addition
of galactose to carbohydrate chain terminating
in N-acetylglucosamine -no free galactose
therefore enzyme will bind to
N-acetylglucosamine but not release
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Hybridization
White-tailed deer x Mule deer Lions x
Tigers Foxes x Coyotes Coyotes x Wolves Dogs
x . Black bears x Polar bears x Grizzly
bears - All offpring generally
fertile -
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Hybridization
Parthenogenesis virgin birth
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Intracytoplasmic sperm injection (ICSI)
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Parthenogenesis Spontaneous 2nd polar body is
not released. Females are heterogametic sex
therefore all offspring are males. Ova x or
y Sperm x Normal female XY
xx male yy dies
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Blue-spotted salamander 2n Ambystoma laterale
Jefferson salamander 2n Ambystoma jeffersonianum
Ambystoma jeffersonianum/laterale complex 3n
females
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2 Populations Diploid females 2n 28
Triploid females 3n 42 3n formed by
female chromosome from sperm 2n from female
All males are diploid 2n 28
Oogenesis Chromosomes of 3n female undergo an
additional division - endomitosis 3n
42 84 After 2 meiotic divisions,
ova with 42 chromosomes are produced 3n
84 3n 42
3 n 42 meiosis I meiosis II
Sperm stimulate fertilization but do not
contribute chromosomes.
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Egg Sperm Zygote 3n (F) n (M/F)
3n (F) Gynogenesis Male genome
not incorporated
3n (F) n (M/F) 3n (M/F) Hybridogenesis Dipl
oid egg formed by elimination of one F
genome then incorporation of male genome
3n (F) n (M/F) 4n (M/F) Tetraploid Incorpor
ation of male genome
Gynogenesis dependent upon water
temperature _at_ 15o C hybridogenesis _at_ 6o
C gynogenesis
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Gray Treefrog Hyla versicolor 4n
Copes Gray Treefrog Hyla chrysoscelis 2n
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Hyla versicolor 4n
Hyla chrysoscelis 2n
East/West lineages
Northern distribution
Normal lineage 3
Separate Tetraploid Lineages
Cold shock after Meiosis II but before 1st
Mitosis 2n 4n
2n x 4n 3n Die or are
sterile
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Gynogenesis
Androgenesis
Genetic marker BL/10 Black Strain SJL
Albino
Blocks cytokinesis by inhibiting actin
polymerization and thus microfilament formation
Hoppe and Illmensee PNAS (1977)
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Regulatory Mechanisms to Date

• Gene amplification (vs. gene replication)
• Hormones (changing concentrations /-
  feedback)
• Hormone receptors (presence/absence/concentrati
  ons)
• Up/down regulation of gene expression
• Gap junctions (cAMP)
• Allosteric regulation of enzyme activity
• Paracrine control (hormones/gap junctions)
• Stabilization of mRNAs
• Turnover rates (half life)
• Presynthesis of mRNA (transcription) and
  storage
• for later translation (unequal
  positional storage)

• Antibodies 1st Ab, 2nd Ab, complexed with
  enzymes,
• fluorescent dyes Monoclonal antibodies
• Fluorescent molecules as substrates (Ca, PKC
  inhibitor)
• Selective Inhibitors Cycloheximide,
  Actinomycin D,
• Cytochalasin B