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Introduction
Spermatogenesis
Oogenesis
Fertilization
Primordial Germ Cells
세포생물학 2 (임현정)
May 29, 2009
Introduction
발생학 (Developmental Biology):
Integration of different levels of biology
1.
Cell
2.
Cell-to-cell communication
3.
Tissue
4.
Organ
5.
Multi-organ system
6.
Interaction with the surrounding
environment
Questions of developmental biology
1.
The question of differentiation
2.
The question of morphogenesis
3.
The question of growth
4.
The question of reproduction
5.
The question of evolution
6.
The question of environmental
integration
Common features of development
1. Genomic equivalence – Cloning of animals
Germ cells (germ line)
Somatic cells
한 개체 내 모든 세포들은 대개의 경우 같다. 그러나 세포들이
다른 이유는 genome 상에서 활성화되어 발현되는 유전자들이
다르기 때문
Cloning: 이미 분화된 한 somatic cell의 핵이 개체 발생을 support
한다는 사실은 모든 DNA가 그대로 있음을 증명
(이것이 genomic equivalence)
2. Behavior of chromosomes during meiosis
4 chromatids
Crossing-over between chromatids
3. Gametogenesis (Germ cell 형성과정)
Gamete
Oogenesis
Spermatogenesis
4. Early development
Gastrulation: formation of three germ layers
Endoderm
Mesoderm
Ectoderm
5. Morphogenesis
Introduction - Key points
1. The main processes in animal development are regional specification, cell differentiation,
morphogenesis, and growth.
2. Whole animal cloning experiments show that the full set of genes is retained by somatic
cells. Development therefore involves the control of gene expression.
3. Gametes arise from cells of the germ line by meiosis.
4. Events at the earliest stages of development involve components preformed in the egg and
so depend on the genome of the mother.
5. Animal development normally involves an early cleavage stage leading to the formation of a
blastula or blastoderm.
6. This early cleavage stage is followed by a phase of morphogenetic movements called
gastrulation during which the three germ layers; ectoderm, mesoderm, and endoderm and
formed.
Spermatogenesis
Key points to remember in Spermatogenesis
1.
Seminiferous ubule의 구조
2.
Spermatogonium의 운명
3.
GDNF
4.
Syncytium
5.
Spermiogenesis
6.
Spermiation
7.
Male infertility
Tunica albuginea: a dense layer of collagen fiber-rich connective tissue
Efferent ductules: sperm transport to epididymis
About 800 seminiferous tubules  rete testis  efferent ductules
Primordial germ cells (PGCs)
Spermatogonium (stem cell)
Type A1
Type A1
Type A2
Type A3
Type A3
Type A4
Type A4
Type B
(1) Self-renewal로 또다른 Type A4 만듬
(2) Cell death (apoptosis)
(3) Differentiation into intermediate spermatogonium
and then divide to form two Type B spermatogonia
Type B
Primary
spermatocyte
Primary
spermatocyte
:enter meiosis
Self-renewal: Type A1은 계속적으로 분열하는 stem cell로 계속적인 germ cell의 pool을 유지한다
(다른 type들도 stem cell일 수 있는 가능성이 있다)
Spermatogonia
Glial cell-derived
neurotrophic factor
(GDNF)
Sertoli cell이 분비
양이 적으면 spermatogonia가 분화하여 spermatocyte로 가고
양이 많으면 spermatoginia가 더 많이 self-renewal 한다
1. Incomplete cytokinesis
•
•
•
•
Spermatogonium 시기부터 세포끼리 cytoplasmic bridge (~1 mm) 로 연결되어 있다. 이는
불완전한 cytokinesis 때문.
Syncytium
Spermatogenesis 동안 세포들이 연결되어 있어 물질이동도 가능
Synchronized development 가능
2. Length of one cycle
•
One round of spermatogenesis: 65 days in humans, ~4 weeks in mice
3. Spermiogenesis
•
One round of spermatogenesis: 65 days in humans, ~4 weeks in mice
4. Spermiation
•
The process by which mature sperm are released from the Sertoli cell into the lumen of
the seminiferous tubule
* Spermiogenesis: The differentiation of the sperm
The process by which spermatids mature into sperm cells
Sertoli cells: support germ cells by providing environment for germ cells to
develop and mature. Sertoli cells also produce hormones such as estorogen and
inhibin. Only somatic cells in the testis.
Leydig cells: located adjacent to seminiferous tubules and they produce
testosterone.
Sertoli-cell-only syndrome
Maturation arrest
Azoospermia: No sperm at all. The ejaculate is completely devoid of sperm.
Oligospermia: (literally, few sperm) There are some sperm, but fewer than normal.
* Overcoming male infertility
1. Assisted reproductive technology (ART)
In vitro fertilization (IVF)
Intracytoplasmic sperm injection (ICSI)
2. In the cases of germ cell depletion or spermatogenic maturation
arrest, spermatogonial transfer could be a therapeutic option.
Oogenesis
Key points to remember in Oogenesis
1.
Developmental arrest
2.
Meiosis: when does it occur?
3.
Hormones and the cycle
4.
Sexual dimorphism in mammalian meiosis
• A human fetus는 2-7개월 사이 증식한 germ cell의 숫자가 7백만 개에 달함
• 이 중 대부분이 atretic death
• 여성에서 성숙되는 난자는 대충 400여 개!
All the follicles and oocytes that an adult female will ever have are present in the ovary at birth (primordial).
A few follicles are activated by FSH from the pituitary during each cycle, progressing from primary to tertiary stage.
Granulosa and thecal cells of follicles are major endocrine source for subsequent implantation.
Prophase
Metaphase
Anaphase
Telophase
Ovulation
Meiosis I
Meiosis II
2n
2n
2n
1n
1n
2n
2n
1n
Arrest
4n
diploid=2n
Fertilization
4n
Arrest
Germinal
Vescile
Germinal
Vesicle (GV)
Germinal Vesicle
Breakdown
(GVBD)
1st Polar Body
(PB)
Completion of
Meiosis II
Figure 19.22 Meiosis in the Mouse Oocyte
tubulin
Figure 19.29(1) The Ovarian Follicle of Mammals
포유류의 배란
1. Copulation (교미)에 의한 배란: 뇌하수체의 gonadotropin 분비 자극으로
2. Periodic ovulation: 설치류의 estrus cycle, 사람의 menstrual cycle
GnRH
Hypothalamus
FHS/LH
Pituitary
Progesterone/estrogen
Ovary
Figure 19.30(1) The Human Menstrual Cycle
This shows the cumulus oophorus (CO), zona pellucida (Z) and
oocyte (Oo) within. Note the nucleus and prominent nucleolus
within the oocyte (Oo).
The corpus luteum
The corpus luteum is the enlarged remnant of the secondary (Graafian) follicle following
ovulation (the release of the oocyte). Note the vacuolization (white circles) associated with
the production of lipid soluable steroid hormones (progesterone and estrogens).
Fertilization
Key points to remember in Fertilization
1.
Internal and external fertilization
2.
Recoginition of sperm and egg
3.
Species-specificity
4.
Prevention of polyspermy
The human infant preformed in the sperm,
as depicted by Nicholas Hartsoeker (1694)
Sperm의 세 가지 조건
The Structure of the Gametes
The modification of a germ cell into a mammalian sperm
Haploid nucleus
A propulsion system
A sac of enzymes
The modification of a germ cell into a mammalian sperm
Mature bull sperm
Proacrosin promoter + GFP
GFP staining
DNA
Tubulin
Mitochondria
The motile apparatus of the sperm
motor protein
11개
13개
Central axoneme and the external fibers
9 + 2 arrangement
The motile apparatus of the sperm (II)
* Sperm capacitation: The sperm released during ejaculation are able to move, but they do not
have the capacity to bind to and fertilize an egg. Capacitation occurs when the sperm is inside
the female reproductive tract for a certain period of time.
Structure of sea urchin egg at fertilization
(glycoproteins)
* Accumulation of materials
(egg cortex, actin 多)
Proteins
Ribosomes and tRNA
mRNA
Morphogenetic factors
Protective chemicals
Stages of egg maturation at the time of sperm entry
4n
2n
n
Recognition of Egg and Sperm
1.
The chemoattraction of the sperm to the egg by soluble molecules secreted by egg
2.
The exocytosis of the acrosomal vesicle to release its enzymes
3.
The bindng of the sperm to the extracellular envelope (vitelline envelope or zona pellucida) of the
egg
4.
The passage of the sperm through this extracellular envelope
5.
Fusion of egg and sperm cell membranes
(2 & 3 could be reversed in some species)
Resact in sea urchin: a chemotactic molecule (14-a.a.)
endows species-specificity
also a sperm-activating peptide
7.8 Summary of events leading to fusion of egg & sperm plasma membranes in the sea urchin
(1)
before
20 sec after injection
40 sec after injection
90 sec after injection
Sperm chemotaxis in the sea urchin Arbacia punctulata. 1 nl of a 10-nM solution of resact is injected
into a 20-ul drop of sperm suspension.
Acrosome reaction in sea urchin sperm
Fusion of the acrosomal vesicle with the sperm cell membrane
The extension of the acrosomal process
Species-specific binding of acrosomal process to egg surface in sea urchins
Bindin: an acrosomal protein mediating egg recognition
dejellied eggs
The Prevention of Polyspermy
The fast block to polyspermy
Change in the electric potential of the egg cell membrane (-70 mV
This is caused by a small influx of sodium ions into the egg.
+20 mV)
The slow block to polyspermy (Cortical granule reaction)
1)
Release of cortical granule serine protease: This enzyme clips off the binding
receptors and any sperm attached to them.
2)
Mucopolysaccharides produce osmotic gradient that causes water to rush into the
space between cell membrane and the vitelline envelope (fertilization envelopes)
3)
Peroxidase: zona hardening
Membrane Potential of Sea Urchin Eggs Before and After Fertilization
Membrane potential of sea urchin eggs
before and after fertilization.
Decreased Na+ concentration in water
increases polyspermy.
Formation of the Fertilization Envelope and Removal of Excess Sperm
Cortical Granule Exocytosis
Schematic diagram showing the events leading to the formation of the fertilization envelope and the hyaline layer.
As cortical granules undergo exocytosis, they release proteases that cleave the proteins linking the vitelline
envelope to the cell membrane. Mucopolysaccharides released by the cortical granules form an osmotic gradient,
thereby causing water to enter and swell the space between the vitelline envelope and the plasma membrane.
Other enzymes released from the cortical granules harden the vitelline envelope (now the fertilization envelope)
and release sperm bound to it.
The Activation of Egg Metabolism
Wave of Calcium Release across Sea Urchin Eggs During Fertilization
Wave of Ca2+ released across a sea urchin egg during fertilization. The egg is preloaded with a dye
that fluoresces when it binds Ca2+. When a sperm fuses with the egg, a wave of calcium release is
seen, beginning at the site of sperm entry and propagating across the egg. The wave takes 30 seconds
to traverse the egg.
Postulated Pathway of Egg Activation in the Sea Urchin
Mammalian Fertilization
Structure of a mammalian egg
Ovulated cumulus-oocyte complex b(COC)
Denudation
Polar body
Zona pellucida (ZP): 투명대
7.30 Sperm-zona binding
Sperm-zona binding in a mammalin egg
(A) Possible model of proteins involved in mouse sperm-egg adhesion. First the sperm binds weakly but
specifically to a ligand protein secreted by the oviduct and coating the zona pellucida. The sperm surface
protein SED1 then binds to the ZP complex on the zona. Sperm galactosyltransferase (GalT) crosslinks
tightly and specifically to N-acetylglucosamine residues on ZP3. The clustering of GalT proteins in the
sperm cell membrane activates G proteins that open calcium channels and initiate the acrosome reaction.
(B) Electron micrograph showing sperm-zona binding in the golden hamster.
7.31 Mouse zona protein 3 binds sperm
Mouse zona proteins 3 (ZP3) binds sperm. (A) Inhibition assay showing a specific decrease of mouse sperm
binding to zonae pellucidae. It appears from this assay that purified ZP3 can bind the sperm and prevent the
sperm from binding to the zona. The assay also illustrates the importance of the carbohydrate portion of ZP3
to the binding reaction. (B) Radioactively labeled ZP3 binds to capacitated mouse sperm.
7.8 Summary of events leading to fusion of egg & sperm plasma membranes in the mouse (2)
* Gamete binding and recognition in mammals
1. Induction of the mammalian acrosome reaction by ZP3
Galactosyltransferase-I: cross-linking sperm ZP3 receptors to ZP3, activation of
Ca++ channel causing exocytosis of the acrosomal vesicles
2.
Traversing the zona pellucida
Exocytosis causes release of various proteases
Secondary binding mediated by ZP2
Acrosome reaction in hamster sperm
acrosomal vesicles
Gamete Fusion
Entry of Sperm into Golden Hamster Egg
Fusion of the Genetic Material
Pronuclear Movements During Human Fertilization
microtubules
sperm
15 hr
sperm tail
unfertilized
The Nonequivalence of Mammalian Pronuclei
Six main techniques of Assisted Reproductive Technology (ART)
• In vitro fertilization (IVF): Your eggs are combined with your partner's sperm in a dish in a
laboratory. Once fertilization occurs, the resulting embryos are placed in your uterus.
• Intracytoplasmic sperm injection (ICSI): Your eggs are combined with one of your
partner's sperm — rather than with many, as in IVF — in a dish in a lab. Once fertilization
occurs, the resulting embryo is placed in your uterus.
• Gamete intrafallopian transfer (GIFT): Your eggs are combined with your partner's sperm
in a dish in a lab, then surgically injected into your fallopian tubes using a laparoscope (a
fiber-thin tube). Fertilization happens inside your body and the embryo implants naturally.
• Zygote intrafallopian transfer (ZIFT): As with GIFT, your eggs are mixed with your partner's
sperm in a dish in a lab, then surgically placed in your fallopian tubes. But, as with IVF, your
doctor will wait until fertilization occurs to place your embryos inside you.
• Donor egg or embryo: If you're unable to conceive using your own eggs, an egg donated
by another woman is mixed with your partner's sperm and the resulting embryo is
implanted in your uterus. This procedure can also be done with a donated embryo.
• Surrogacy (or use of a gestational carrier): Another woman carries your embryo, or a
donor embryo, to term and gives the baby to you after birth.
Primordial Germ Cells
1. All gametes arise from PGCs.
2. Frogs, nematodes, flies, etc.: PGCs are autonomously specified by cytoplasmic determinants
(Germ Plasm) during cleavage.
3. Mammals, etc.: PGCs are specified by interactions among neighboring cells.
Chromosome diminution (roundworm Parascaris)
이로 인해 많은 유전자가 사라지나 germ cell이
될 세포에서는 모든 유전정보가 유지된다
원심분리로 인해 germ plasm이
두 개의 세포에 나뉘게 되어 두
개의 stem cell이 생긴다
“Germ plasm”: 특정 RNA나 protein이 이를 specify 한다
19.3 The pole plasm of Drosophila
The pole plasm of Drosophila
19.6 Germ plasm at the vegetal pole of frog embryos
Germ plasm at the vegetal pole of frog embryos
Xcat2 mRNA의 localization
Day 7 mouse embryo
포유류에서는 germ plasm이
없다. 그대신 embryo에서
유도되어 만들어진다.
(요막)
(Epiblast)
19.7 Specification and migration of mammalian primordial germ cells
Day 7 mouse embryo
Morphogens
Extraembryonic ectoderm과 epiblast junction에서
germ cell들이 유도되어 나온다.
Posterior epiblast 부분으로 PGC가 들어온다.
fragilis (+)
stella (+)
blimp1 (+)
Expression of Oct4 mRNA correlates with totipotency and ability to form germ cells
Inner cell mass
Spermatogonia
Posterior
Epiblast (8.5)
Oogonia
Migrating
PGCs (10.5)
19.14 Primoridal germ cell migration in the mouse (Part 1)
Hindgut
19.14 Primoridal germ cell migration in the mouse (Part 2)