EMBRYOLOGY LABORATORY
Exercise 2, 3 & 4.1
FERTILIZATION & MATURATION STAGE | EARLY DEVELOPMENT | NEURULATION IN FROGS
FERTILIZATION & MATURATION
Ascaris (Microlecithal Egg)
- Empty Shell
1. Unfertilized Egg
2. (1) Sperm Penetration Stage
- Primary Oocyte
3. Maturation stage
st
- 1 Polar Body Formation
- 2nd Polar Body Formation
+ Secondary Oocyte
- (2) Pronuclear Stage
+ Ootid
+ Female Nucleus
+ Male Nucleus
- (3) Fusion Stage
+ Ovum
- (4) Early Cleavage
+ Embryo (Zygote)
+ PMAT
EARLY DEVELOPMENT
Starfish (Microlecithal Egg)
1. Unfertilized Egg
2. Fertilized Egg
3. Cleavage Division Stage
- Early Cleavage
+ 2 cell stage
+ 4 cell stage
+ 8 cell stage
- Late Cleavage
+ 16 Cell Stage
+ 32 Cell Stage (morula)
4. Blastula
- Blastocoel
- Blastoderm
- Fertilization Membrane
- Invaginating Cells
5. Gastrula
- Archenteron / Gastrocoel
- Enterocoelic pouch
- Blastopore
- Vesicles
- Mesenchyme
- Bipinnaria Larva
FERTILIZATION & MATURATION STAGE
fertilization direct interaction and fusion of two germinal cells
o one egg + one spermatozoan
o result: initiation of cleavage, gastrulation and the species-specific
developmental program that characterizes each organism
OUTLINE
Ascaris (2n = 4) parasitic nematode in the intestine of some mammals
EARLY DEVELOPMENT
o Well-developed reproductive system
Frog (Medialecithal Egg) Chick (Macrolecithal Egg) Separate sexes
1. Cleavage Division Stage
18 Hr Chick Ovary coiled and Thread-like, enlarges at the posterior as uterus
- Nucleus
- Fertilization Membrane - Area Opaca Vagina fusion of uteri of two branches
- Early Cleavage - Area Pellucida Vulva (Genital Pore) ending of Vagina
- Epiblast
+ 2 cell stage o Fertilization - @ the end of uterus near the oviduct
- Hypoblast
+ 4 cell stage o Ascaris eggs Transparent
+ 8 cell stage - Primitive streak
+ Primitive Groove
+ 16 cell stage first maturation division of oogenesis & ovulation = secondary oocyte (fertilization)
+ Primitive Ridges
- Morula (32) complete oogenesis
2. Blastula + Primitive Plate
+ Primitive Pit
- Animal Pole no Fertilization - degenerate
+ Hensens Node
- Vegetal Pole second maturation division
- Blastocoel @ metaphase fertilization continue
- Micromeres
- Macromeres
- Blastoderm Fertilization
- Fertilization Membrane o sperm entrance continued meiotic division
3. Gastrula o presence of sperm at the egg surface triggers development
- Gastrocoel / Archenteron o sperm penetrates the egg prior to the completion or even the start of meiosis
- Blastopore Stage of Development o sperm head stays in the cytoplasm of the egg until female nucleus is formed
- Lip of the Blastopore - 2 cell stage - o male and female chromosomes are joined fertilization completed
+ Dorsal Lip
- 4 cell stage - o Formation of zygote
+ Ventral Lip - 8 Cell Stage -
- Yolk Plug - 16 Cell Stage -
- 3 Germ Layers (M, En, Ec) Stages of Maturation
- 32 Cell Stage -
- Blastocoel 1. Sperm Penetration Stage
- Fertilization Membrane Maturation Stage
4. Neurula 2. Pronuclear Stage
Neural Plate Stage 3. Fusion Stage
+ Neural Plate
Stage of Maturation 4. Early Cleavage
+ Presumptive Epidermis - Cleavage -
+ Notochord - Morula -
+ Mesoderm - Blastula - Fertilization of a Microlecithal Egg
+ Endoderm
- Gastrula -
Neural Fold Stage - Neurula -
+ Neural Fold Unfertilized egg - extensive membranes observed in
+ Neural Groove fertilized eggs are absent here
+ Hypochondral o Vacuolated cytoplasm bubbles
o Inconspicuous nuclei
Neural Tube Stage
+ Neural Crest Cells o Thin cell membranes
+ Neurocoel o Elongated and larger than fertile egg
+ Mesoderm (Epi, Meso, o Thin shelled
Hypomere)
o Shell ranges from irregular mammillations to relatively smooth layer
NexCab, JoFred, et. al (pictures). 2017
 Sperm Penetration Stage
o Stage of development: Primary Oocyte Second polar body formation
Have a vacuolated cytoplasm o Second Polar Body formed when a secondary oocyte
Inconspicuous nuclei extrudes one half of its longitudinal split chromosomes
Thin cell membrane Separation of sister chromatids
After fertilization, they become surrounded by a looks exactly like a first polar body
fertilization membrane and thick outer shell 2 Criteria to classify 2nd Polar Body:
(Chitinous) 2 black streaks observed & it is the inner streak
o presence of the sperm head inside the egg Only 1 streak appears, but the female pronucleus is already formed
Sperm heads small, dense and triangular bodies nd nd
o Ootid formed after the 2 meiotic division along w/ 2 polar body
o Nucleus with a different shape of consistency no clear distinction between an ootid and an ovum
o Presence of bivalents male and the female pronuclei will only fuse if the egg cell is already
functional (Ovum)

Maturation stage

First polar body formation

o Ascaris 2n = 4
o separation of the members of the homologous pair =
two groups of 4 chromosomes
o Chromosomes appear as bread-like structures
o Tetrads that will be part of the polar body will be found
Pronuclear stage
in the periphery of the primary oocyte
o Completion of maturation process of oocyte before the actual fusion
o first or Primary Polar Body appears like a small spherical structure attached
o male pronucleus appears as a dense body near the center of the egg
on the membrane of the oocyte and enveloped by the cytoplasm.
o perivitelline space (between the egg and the fertilization membrane) is
can also be observed on the fertilization membrane (inner layer of the
filled with fluid
chitinous layer) as a black streak
o sperm head pronuclei : HAPLOID sperm head & some cytoplasm from the
spermatozoan are drawn into the cytoplasm of the egg; nucleus swells; its Fusion stage
o Female & male pronuclei fuse following their migration towards each other.
centriole generates an array of microtubules
male pronucleus nucleus of the sperm after it has penetrated the produces the diploid zygote nucleus
o membranes of the male and female pronuclei are fusing.
cytoplasm of the ovum and enlarges
o female pronucleus nucleus of the ovum; product of the second meiotic o In some cases, an array of microtubules can be observed near the centrioles
division of the female and male nuclei terminates the process of fertilization

NexCab, JoFred, et. al (pictures). 2017

o Fusion of male and female pronuclei EARLY DEVELOPMENT
nd st
Presence of 2 polar bodies (inner-2 polar body; outer-1 polar body) or 1
nd st
polar body (2 polar body near the outer covering; 1 polar body already development of an organism increase in mass and elaboration of structures
extruded from the shell) o cell activity increase in cell number (due to mitosis), enlargement of cells,
o Perivitelline space space between the egg and the fertilization membrane migration of cells, differentiation and specialization of cells
o Stage of development: Ovum
Cleavage mechanism for cloning the zygote genome generated at fertilization
following male and female pronuclear fusion
o occurs right after fertilization
o zygote Blastula : series of mitotic cell division: segmentation or cleavage
o Holoblastic Cleavage cell division involves the whole egg
Blastula consists of a hollow ball of cells
o Gastrulation first time many organisms begin to express the new embryonic
genome that was formed at fertilization
Early cleavage
o Cleavage and later developmental processes occur while the egg is still inside Classification of Eggs according to the relative amount of yolk which they contain:
the uterus o microlecithal or oligolecithal with little yolk
o Stage of Development: Zygote
Echinoderms, coelenterates, amphioxus & mammals except monotremes
Diploid o medialecithal or mesolecithal with moderate amount of yolk
Large Chromosomes Eggs of annelid, mollusks, lampreys, lungfishes and ampbihibians
o Holoblastic Entire cell dividing o megalecithal or macrolecithal with large amount of yolk
Arthropods, hag fishes, bony fishes, reptiles, birds and monotremes

Classification of Eggs according to the distribution of yolk:

o Isolecithal even distribution of yolk
a microlecithal egg is isolecithal
o telocithal most of its yolk concentrated on one pole the vegetal pole
amphibians are moderately telolecithal; birds are highly telolecithal
Prophase o centrolecithal yolk is concentrated in the interior of the egg, and the
cytoplasm is distributed as a thin coat on the external surface
In arthropods, especially insects

Cleavage

Cleavage not simply multiplication of cells but produces cells which, eventually,
become arranged distinctly
Metaphase Anaphase o cleavage cells or blastomeres
o due to the predetermined pattern influenced by the amount and distribution
of the yolk among vertebrates
yolk very important factor in the cleavage formation because its
presence may inhibit or slow down cell division
o Cleavage Formation
Holoblastic in eggs with small amount of yolk or none at all (alecithal)
and is equally distributed; blastomeres of equal size
Teloblastic medium amount of yolk (mesolecithal or medialecithal), the
Telophase yolk are pulled to its lower position (the vegetal pole)
macromeres bigger and fewer blastomeres @ vegetal pole
micromeres many small blastomeres @ animal pole

NexCab, JoFred, et. al (pictures). 2017

 Microlecithal egg (Starfish)
o Unfertilized egg inactivated ovum of the starfish
prominent prematuration nucleus germinal
vesicle w/ a distinct nucleolus
viteline membrane difficult to identify because
of its close adherence to the egg
a primary membrane formed by the egg
o Fertilized egg
elevated vitelline membrane (aka fertilization membrane)
perivitelline space a space formed by the separation of fertilization
membrane from the egg
germinal vesicle of the fertilized egg has broken down in preparation for
the maturation process
sperm head densely staining body within the egg cortex
o Cleavage division stage
The starfish egg exhibits holobastic radial cleavage

Blastula

Blastula a hollow ball of cells resulting from the successive cleavages

o Blastocoel centrally located cavity, the walls of which consist an epithelial
layer of cells
o Blastoderm layer of the blastomeres arranged at the side of the embryo and
enclosing the blastocoels

Fertilization membrane thin membrane surrounding the blastula

o secondary membrane because it is not secreted by the egg but by follicle
cells in the ovary
o (medialecithal egg) Chorion + Viteline membrane

Microlecithal egg (Starfish)

o Blastocoel not readily identifiable because the cells at the back part of the
section is still visible but the organization of the blastomeres at the external
Medialecithal egg (Frog) surface of the embryo is prominent
o Amphibian egg mesolecithal, having an intermediate amount of yolk
larger than that of the starfish due to the larger quantity of yolk
o 2 distinct divisions:
dark pigmented hemisphere (the animal hemisphere)
lightly or unpigmented hemisphere (the vegetal hemisphere)
o pigement granules reside in the cortical cytoplasm of the egg within the
plasma membrane and are more abundant in the animal hemisphere
o yolk distributed throughout the cytoplasm of the egg but is much more
concentrated toward the vegetal pole end of the egg

NexCab, JoFred, et. al (pictures). 2017

 Medialecithal egg (Frog) Microlecithal egg (starfish)
o Animal pole region where o After the formation of a relatively large blastocoels
numerous smaller cells are found o first sign: starfish embryo becomes flattened at the vegetal pole (the initial
micromeres migration and differentiation of cells
o Vegetal pole region where fewer o cells will then invaginate within the blastocoel formation of internal layers
but bigger cells are found give rise to the endoderm and mesoderm germ layers
Macromeres o ectoderm cells which remain on the outer surface of the gastrula
o Vesicles diverticula or lateral pouches at the apical region of the gastrocoel
become separated from the gastrocoel and give rise to the many
mesodermal structures of the animal
aka Coelomic Sacs
cut at animal
o Mesenchyme large, stellate cells which are mesodermal in origin
pole only
Scattered between the ectoderm and gastrocoel
arise by budding off from the outer walls of the gastrocoel
o Bipinnaria larva larva of starfish formed at the end of the gastrula stage
presence of the cilia covering the entire ectodermal surface of the
embryo; principal locomotors organs of the larva
Gastrula

Gastrulation stage characterized by morphogenetic movement and well-ordered

rearrangement of cells after the cleavage stage and blastula stages
o gastrula that is formed = embryo
o occurs after invagination happens at the vegetal pole
o 3 kinds of cell movements
Involution in-turning or inward movement of the outer layer of cells so
that it spreads out under the internal surface of the outer layer of cells
Invagination - creates the beginning of the archenteron
Epiboly continues as the germ ring advances over the exposed yolk cells
o Groupings of cells of the same developmental potentialities occur
o formation of the 3 germ layers: ectoderm, mesoderm and the endoderm

different kinds of gastrulation

o (microlecithal egg) simple gastrulation: in pocketing of the blastula with the
formation of a double-layered cup
o (mesolecithal egg) formation of a dorsal blastoporal lip w/in the grey crescent
o (macrolecithal) chick and the placental mammals: follow the same general
pattern that is the formation of a primitive streak Medialecithal egg
o dorsal lip of the blastopore - upper margin of the curved fold; more defined
Involution first occurred at the dorsal lip near the more rapidly dividing
Gastrula 2 layered embryo from the ectodermal movements of cell
animal pole
The blastopore is now surrounded by dorsal, lateral and ventral lips
gastrocoel or archenteron (future primitive gut) - cavity created by the
o yolk plug area of yolk that can only be seen within the confines of the
invagination of cells digestive tract
blastopore; endodermal in origin
o blastopore opening at the vegetal pole end; marks the posterior end of the
o endoderm and chordamesoderm from the expanding layer of cells which
embryo and becomes the future anus
forms the roof of the archenteron
mouth of the in-pocketing of cells
o ectoderm cells on the outer surface of the embryo
The length of the gastrocoel indicates the specific stage of gastrula whether early, middle or o mesoderm soon develops between the ectoderm and the endoderm.
late. Early gastrula exhibits a short gastrocoel whereas in late gastrula, the gastrocoel o Blastocoel remnant of this may be seen.
extends over half the length of the embryo and its apex may be seen a thin-walled vesicle o Fertilization membrane thin membrane that envelopes the gastrula

NexCab, JoFred, et. al (pictures). 2017

 o Hensens node elevation immediately anterior to the primitive pit
represented by the streaks that bound the primitive pit
where the presumptive notochordal cells migrate
Syn: Primitive knot

Megalecithal egg (Chick)

o Gastrulation starts with a thickening of the cells in the posterior portion of the
area pellucida
cells inside the blastodisc split and proliferate into the blastocoels
hypoblasts cells that splitted off
epiblasts remaining cells on top
Epiboly and involution holds true also in megalectihal eggs, but the manner in NEURULATION
which they are accomplished is diffrerent from the medialecithal eggs.
o Area opaca the darker, outer, peripheral area seen in the specimens (due to
Neural Plate Stage earliest stage
the presence of yolk beneath it)
o Neural Plate (aka medullary plate) region of embryonic ectodermal cells
o Area pellucida the clearer, central area in the specimen.
(neuroectoderm), that lie directly above the notochord
separated from the yolk by the segmentation cavity
o Presumptive epidermis remainder of the ectoderm after the formation of
o Primitive streak the thickened part of the blastoderm
the medullary plate
appears as a long streak in the center part of the specimen
Forerunner of the outer layer of the integument
brought by the convergence of its surface layer toward the midline in the
o Notochord oval mass directly beneath the neural plate and fr the mesoderm
posterior half of the area pellucida.
Posibbly fr the dorsal region of the archenteron roof
homologous to the epiboly in amphibians
Provides skeletal support during early development
The cells that sink inside this thickened area forms a layer of cells
Temporary
between the epiblasts and the hypoblasts
o Mesoderm middle germ layer formed in the embryo during gastrulation
ectoderm remaining cells on the surface after the immigration
Found on each side of the notochord and dorsal to the archenteron roof
endoderm innermost layer of cells near the blastocoel which has
3 regions of mesoderm in early organogenesis:
contributed some cells to the middle layer of sunken cells to form the
Epimere dorsal mesoderm; forms somites
mesoderm
Mesomere intermediate mesoderm; nephrotome; forms excretory
o Primitive groove the central furrow
o Primitive ridges the thickened margins on both sides of the primitive groove system
o Primitive plate thickening at the caudal end of the streak Hypomere lateral plate mesoderm; delaminates into the inner
o Primitive pit found at the cranial most end of the streak. This appears in the splanchnic and outer somatic mesoderm
slide as a light area flanked by darkly-stained small streaks. o Endoderm large yolk-ladden cells that surrounds the gastrocoel

NexCab, JoFred, et. al (pictures). 2017

 Mesomere stalk-like connection b/n the epimere and hypomere
Forms: Pronephros and Metanephros
Aka nephrotome, nephromere
Hypomere posterior to mesomere
Split into 2:
- Outer somatic mesoderm
- Interior splanchnic mesoderm
Coelom cavity b/n the 2 divisions

Neural Fold Stage neural folds are prominently elevated

o Neural fold portion of the neural plate that has a depression and a laterally
elevated margin
o Neural groove depression formed in the midline as the neural folds meet at
the mid-dorsal region of the embryo
Gives rise to the neurocoel

Neural tube stage neural folds have already fused at the mid-dorsal region
forming a neural tube with neurocoel
o Neural crest cells group of cells found at the edges of the neural plate
Lying above the neural tube after its formation
Migrate throughout the embryo to form ganglia, symphatetic nervous
system, pigment cells, parts of gill arches, etc
o Mesoderm
Epimere segmented mesodermal blocks @ either side of the notochord
Beneath the epidermal ectoderm
Forms:
- Sclerotome axial part of the skeleton
- myotome muscle
- dermatome dermal layer of the skin

NexCab, JoFred, et. al (pictures). 2017