1. Introduction Spermatogenesis Oogenesis Fertilization 세포생물학 2 (임현정)
March 12, 2010

2. Introduction 발생학 (Developmental Biology):
Integration of different levels of biology
Cell
Cell-to-cell communication
Tissue
Organ
Multi-organ system
Interaction with the surrounding environment
Questions of developmental biology
The question of differentiation
The question of morphogenesis
The question of growth
The question of reproduction
The question of evolution
The question of environmental integration

3. 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)

4. 2. Behavior of chromosomes during meiosis
4 chromatids
Crossing-over between chromatids

5. 3. Gametogenesis (Germ cell 형성과정)
Gamete
Oogenesis
Spermatogenesis

6. 4. Early development
Gastrulation: formation of three germ layers
Endoderm
Mesoderm
Ectoderm

7. 5. Morphogenesis

8. 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.

9. Spermatogenesis Key points to remember in Spermatogenesis
Seminiferous ubule의 구조
Spermatogonium의 운명
GDNF
Syncytium
Spermiogenesis
Spermiation
Male infertility

10. 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

13. devbio8e-fig jpg

14. Spermatogonium (stem cell)
Primordial germ cells (PGCs)
Spermatogonium (stem cell)
Type A1
Type A1
Type A2
Type A3
Type A3
Self-renewal로 또다른 Type A4 만듬
Cell death (apoptosis)
Differentiation into intermediate spermatogonium and then divide to form two Type B spermatogonia
Type A4
Type A4
Type B
Type B
devbio8e-fig jpg
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)

15. Spermatogonia Glial cell-derived neurotrophic factor (GDNF)
devbio8e-fig jpg
Sertoli cell이 분비
양이 적으면 spermatogonia가 분화하여 spermatocyte로 가고
양이 많으면 spermatoginia가 더 많이 self-renewal 한다

16. 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

17. * Spermiogenesis: The differentiation of the sperm
The process by which spermatids mature into sperm cells

19. 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.

20. * Overcoming male infertility
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.

21. Oogenesis Key points to remember in Oogenesis Developmental arrest
Meiosis: when does it occur?
Hormones and the cycle
Sexual dimorphism in mammalian meiosis

22. devbio8e-fig jpg
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.

25. Germinal Vesicle Breakdown
Prophase
Metaphase
Anaphase
Telophase
Meiosis I
Fertilization
Germinal
Vesicle (GV)
Germinal Vesicle Breakdown
(GVBD)
1st Polar Body
(PB)
Ovulation
Arrest
Meiosis II
Completion of

26. Figure 19.22 Meiosis in the Mouse Oocyte
tubulin
Figures\Chapter19\DevBio7e19220.jpg

27. Figure 19.29(1) The Ovarian Follicle of Mammals
Figures\Chapter19\DevBio7e19291.jpg
포유류의 배란
Copulation (교미)에 의한 배란: 뇌하수체의 gonadotropin 분비 자극으로
Periodic ovulation: 설치류의 estrus cycle, 사람의 menstrual cycle
GnRH
Hypothalamus
FHS/LH
Pituitary
Progesterone/estrogen
Ovary

28. Figure 19.30(1) The Human Menstrual Cycle
Figures\Chapter19\DevBio7e19301.jpg

29. This shows the cumulus oophorus (CO), zona pellucida (Z) and oocyte (Oo) within. Note the nucleus and prominent nucleolus within the oocyte (Oo).

30. 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).

31. devbio8e-table jpg

33. Fertilization Key points to remember in Fertilization
Internal and external fertilization
Recoginition of sperm and egg
Species-specificity
Prevention of polyspermy
The human infant preformed in the sperm,
as depicted by Nicholas Hartsoeker (1694)

34. The Structure of the Gametes
Sperm의 세 가지 조건
Haploid nucleus
A propulsion system
A sac of enzymes
The Structure of the Gametes
The modification of a germ cell into a mammalian sperm

35. The modification of a germ cell into a mammalian sperm
Mature bull sperm
Proacrosin promoter + GFP
GFP staining
DNA
Tubulin
Mitochondria

36. The motile apparatus of the sperm
motor protein
11개
13개
Central axoneme and the external fibers
9 + 2 arrangement

37. 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.

38. Structure of sea urchin egg at fertilization
(glycoproteins)
* Accumulation of materials
Proteins
Ribosomes and tRNA
mRNA
Morphogenetic factors
Protective chemicals
(egg cortex, actin 多)

39. Stages of egg maturation at the time of sperm entry

40. Recognition of Egg and Sperm
The chemoattraction of the sperm to the egg by soluble molecules secreted by egg
The exocytosis of the acrosomal vesicle to release its enzymes
The bindng of the sperm to the extracellular envelope (vitelline envelope or zona pellucida) of the egg
The passage of the sperm through this extracellular envelope
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

41. 7.8 Summary of events leading to fusion of egg & sperm plasma membranes in the sea urchin (1)
devbio8e-fig jpg

42. before
20 sec after injection
40 sec after injection
90 sec after injection
devbio8e-fig jpg
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.

43. Acrosome reaction in sea urchin sperm
Fusion of the acrosomal vesicle with the sperm cell membrane
The extension of the acrosomal process

44. Bindin: an acrosomal protein mediating egg recognition
Species-specific binding of acrosomal process to egg surface in sea urchins
Bindin: an acrosomal protein mediating egg recognition
dejellied eggs

45. The Prevention of Polyspermy
The fast block to polyspermy
Change in the electric potential of the egg cell membrane (-70 mV mV)
This is caused by a small influx of sodium ions into the egg.
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.
Mucopolysaccharides produce osmotic gradient that causes water to rush into the space between cell membrane and the vitelline envelope (fertilization envelopes)
Peroxidase: zona hardening

46. Membrane Potential of Sea Urchin Eggs Before and After Fertilization
Decreased Na+ concentration in water increases polyspermy.

47. Formation of the Fertilization Envelope and Removal of Excess Sperm

48. 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.

49. 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.

50. devbio8e-table jpg

51. Postulated Pathway of Egg Activation in the Sea Urchin

52. Mammalian Fertilization
Structure of a mammalian egg
Ovulated cumulus-oocyte complex b(COC)
Denudation
Polar body
Zona pellucida (ZP): 투명대

53. 7.30 Sperm-zona binding Sperm-zona binding in a mammalin egg
devbio8e-fig jpg
(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.

54. 7.31 Mouse zona protein 3 binds sperm
devbio8e-fig jpg
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.

55. 7.8 Summary of events leading to fusion of egg & sperm plasma membranes in the mouse (2)
devbio8e-fig jpg

56. * 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
Traversing the zona pellucida
Exocytosis causes release of various proteases
Secondary binding mediated by ZP2

57. Acrosome reaction in hamster sperm
acrosomal vesicles

58. Gamete Fusion
Entry of Sperm into Golden Hamster Egg

59. Fusion of the Genetic Material
Pronuclear Movements During Human Fertilization
microtubules
sperm
15 hr
sperm tail
unfertilized

60. The Nonequivalence of Mammalian Pronuclei

61. 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.