National Agriculture and Biooriented

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National Agriculture and Bio-oriented Research
OrganizationNational Institute of Livestock and
Grassland Science
kyoto
Tokyo
Takashi (TAKU) Nagai
E-mail taku_at_affrc.go.jp Tel [phone],
Fax [phone]
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Journey of sperm through female genital tracts to
meet ova and create a new life in livestock
animals
Sperm transport in female genital tracts Sperm
trap at ithmus part of oviduct Fertilization at
ampulla part of oviduct Capacitation and
acrosome reaction of sperm Sperm penetration
into ova Early embryo development
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Special prologue with a Japanese traditional
Shin-to festival called TAMA-TORI-SAI
Taking an egg (soul) festival in English

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Artificial insemination
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Functional anatomy of the female
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Animal Science 434 Lectures Wisconsin University
http//www.wisc.edu/ansci_repro/lec/lectures.html
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Sperm ascent in the female tract
No. of spermatozoa
Sheep, Cow 100-1,000 Pig, Horse 1,000-2,000
No. of spermatozoa
Isthmus
Sheep, Cow 1 x 105 Pig, Horse 1 x 109
Phases of sperm transport a) Rapid (15 - 30
min) b) Sustained 6-12 hr) c) Oviductal (at
ovulation, 1-2 hr)
Ampulla
Oocyte
Cumulus cells
Uterus
Follicle
Ovary
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Sperm loss from the female tract 1) Retrograde
flow 2) Phagocytosis by neutrophils
Evolutionary strategies for dealing with
retrograde flow of semen 1) Coagulation of
semen or a plug 2) Concentrated semen 3) Semen
deposited in uterus
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Sperm ascent in the female tract
No. of spermatozoa
Sheep, Cow 100-1,000 Pig, Horse 1,000-2,000
No. of spermatozoa
Isthmus
Sheep, Cow 1 x 105 Pig, Horse 1 x 109
Ampulla
Oocyte
Cumulus cells
Uterus
Follicle
Ovary
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Sperm ascent in the female tract
No. of spermatozoa
Sheep, Cow 100-1,000 Pig, Horse 1,000-2,000
No. of spermatozoa
Isthmus
Sheep, Cow 1 x 105 Pig, Horse 1 x 109
Ampulla
Oocyte
Cumulus cells
Uterus
Follicle
Ovary
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Follicle
Granulosa cells
Theca cells
Basement membrane
Oocyte
Nucleus
Cumulus cells
Oocyte
Zona pellucida
0.1 mm
10-20 mm
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Cumulus cells
Zona pellucida
Oocyte cytoplasm
Nucleus
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Sperm ascent in the female tract
No. of spermatozoa
Sheep, Cow 100-1,000 Pig, Horse 1,000-2,000
No. of spermatozoa
Isthmus
Sheep, Cow 1 x 105 Pig, Horse 1 x 109
Ampulla
Oocyte
Cumulus cells
Uterus
Follicle
Ovary
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In Vitro Fertilization of In Vitro Matured Oocytes
Materials and methods
Ovaries from a slaughterhouse
COCs collection
Sperm capacitation
IVM
Culture Medium
IVF
IVC
At 38.5OC, 5 CO2 in air
Oocyte-sperm coculture
To evaluate the fertilization status
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Structure of sperm head
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Sperm capacitation in vitro a)
Changes in the Acrosome b) Regulation of
Capacitation by Ca2 c) Removal of
de-capacitation factors Epididymal
spermatozoa vs. Ejaculated Spermatozoa
d) Increase of pH and cAMP e)
Hyperactivated motility of spermatozoa
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• Decapacitation Factors
• Protein, glycolipid and/or lipid
• b) Stabilizes the plasma membrane to delay or
  prevent
• capacitation
• c) Can be secreted from the epididymis and/or
  accessory
• glands

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F (un-capacitated)-pattern, bright fluorescence
was detected whole region of sperm head B
(capacitated)-pattern, fluorescence was detected
on sperm head except postacrosomal region AR
(acrosome reacted)-pattern, weak fluorescence was
observed over the sperm head and bright band was
sometimes observed in the equatorial segment.
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Acrosome reaction in vitro a)
Zona-mediated acrosome reaction b) Ca2
uptake of spermatozoa
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Function of Acrosome
Enzymes Hyaluronidase Hyaluronidase
released form acrosome-reacting spermatozoa
is thought to digest the cumulus
matrix. Acrosin Acrosin carried on the
surface of acrosome-reacted spermatozoa is
thought to aid in the passage of spermatozoa
through zona pellucida. Proacrosin
Proacrosin, an inactive form of acrosin, has a
strong zona-binding ability.
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Miclovilli
Fusion of sperm and oocyte
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Functions of the Acrosome Reaction
1) Rendering spermatozoa capable of
penetration through zona pellucida. 2)
Rendering spermatozoa capable of
fusing with egg plasma membrane.
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Possible relationships among sperm capacitation,
acrosome reaction, and hyperacrtivation
Capacitation
Changes in the head plasma membrane
Changes in the tail plasma membrane
Acrosome reaction Zona penetration Sperm-egg
fusion
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Scanning electron micrograph of a golden hamster
spermatozoon about to enter the zona pellucida.
The acrosome ghost (arrow) is seen.
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How miserable spermatozoa are!
But
They are very important to wake up oocytes from
a deep sleep!
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Confocal microscopic image of microtubule
organization during bovine fertilization
GreenAster of microtubules BlueNucleus
(propidium iodide) MMale pronucleus?FFemale
pronucleus
Navara, C.S., First N.L., and Schatten G. (1996)
Proc. Natl. Acad. Sci. USA 93, 5384-5388.
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Hatching of mouse embryo
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Thank you!