EMBRYOLOGY LABORATORY
FERTILIZATION & MATURATION STAGE
Exercise 2, 3 & 4.1 FERTILIZATION & MATURATION STAGE | EARLY DEVELOPMENT | NEURULATION IN FROGS
OUTLINE FERTILIZATION & MATURATION
EARLY DEVELOPMENT
Ascaris (Microlecithal Egg)
Frog (Medialecithal Egg) 1. Cleavage Division Stage - Nucleus - Fertilization Membrane - Early Cleavage + 2 cell stage + 4 cell stage + 8 cell stage + 16 cell stage - Morula (32) 2. Blastula - Animal Pole - Vegetal Pole - Blastocoel - Micromeres - Macromeres - Blastoderm - Fertilization Membrane 3. Gastrula - Gastrocoel / Archenteron - Blastopore - Lip of the Blastopore + Dorsal Lip + Ventral Lip - Yolk Plug - 3 Germ Layers (M, En, Ec) - Blastocoel - Fertilization Membrane 4. Neurula Neural Plate Stage + Neural Plate + Presumptive Epidermis + Notochord + Mesoderm + Endoderm Neural Fold Stage + Neural Fold + Neural Groove + Hypochondral Neural Tube Stage + Neural Crest Cells + Neurocoel + Mesoderm (Epi, Meso, Hypomere)
- 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 – 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
Ascaris (2n = 4) – parasitic nematode in the intestine of some mammals o Well-developed reproductive system Separate sexes Ovary – coiled and Thread-like, enlarges at the posterior as uterus Vagina – fusion of uteri of two branches Vulva (Genital Pore) – ending of Vagina o Fertilization - @ the end of uterus near the oviduct o Ascaris eggs – Transparent
Chick (Macrolecithal Egg) 18 Hr Chick - Area Opaca - Area Pellucida - Epiblast - Hypoblast - Primitive streak + Primitive Groove + Primitive Ridges + Primitive Plate + Primitive Pit + Hensen’s Node
Stage of Development - 2 cell stage - 4 cell stage - 8 Cell Stage - 16 Cell Stage - 32 Cell Stage -
Stage of Maturation - Cleavage - Morula - Blastula - Gastrula - Neurula -
first maturation division of oogenesis & ovulation = secondary oocyte (fertilization) → complete oogenesis no Fertilization - degenerate second maturation division @ metaphase
fertilization – continue
Fertilization o sperm entrance – continued meiotic division o presence of sperm at the egg surface – triggers development o sperm penetrates the egg prior to the completion or even the start of meiosis o sperm head stays in the cytoplasm of the egg until female nucleus is formed o male and female chromosomes are joined – fertilization completed o Formation of zygote
Stages of Maturation 1. Sperm Penetration Stage Maturation Stage 2. Pronuclear Stage 3. Fusion Stage 4. Early Cleavage
Fertilization of a Microlecithal Egg
Unfertilized egg - extensive membranes observed in fertilized eggs are absent here o Vacuolated cytoplasm – bubbles o Inconspicuous nuclei o Thin cell membranes o Elongated and larger than fertile egg o Thin shelled 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 Have a vacuolated cytoplasm Inconspicuous nuclei Thin cell membrane After fertilization, they become surrounded by a fertilization membrane and thick outer shell (Chitinous) o presence of the sperm head inside the egg Sperm heads – small, dense and triangular bodies o Nucleus – with a different shape of consistency o Presence of bivalents
Second polar body formation o Second Polar Body – formed when a secondary oocyte extrudes one half of its longitudinal split chromosomes Separation of sister chromatids looks exactly like a first polar body 2 Criteria to classify 2nd Polar Body: 2 black streaks observed & it is the inner streak Only 1 streak appears, but the female pronucleus is already formed nd nd o Ootid – formed after the 2 meiotic division along w/ 2 polar body no clear distinction between an ootid and an ovum male and the female pronuclei will only fuse if the egg cell is already functional (Ovum)
Pronuclear stage o Completion of maturation process of oocyte before the actual fusion o male pronucleus – appears as a dense body near the center of the egg o perivitelline space – (between the egg and the fertilization membrane) is filled with fluid
Fusion stage o Female & male pronuclei fuse following their migration towards each other. produces the diploid zygote nucleus o membranes of the male and female pronuclei are fusing. o In some cases, an array of microtubules can be observed near the centrioles of the female and male nuclei – terminates the process of fertilization
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 in the periphery of the primary oocyte o first or Primary Polar Body – appears like a small spherical structure attached on the membrane of the oocyte and enveloped by the cytoplasm. can also be observed on the fertilization membrane (inner layer of the 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 centriole generates an array of microtubules male pronucleus – nucleus of the sperm after it has penetrated the cytoplasm of the ovum and enlarges o female pronucleus – nucleus of the ovum; product of the second meiotic division
©NexCab, JoFred, et. al (pictures). 2017
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Fusion of male and female pronuclei 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 extruded from the shell) Perivitelline space – space between the egg and the fertilization membrane Stage of development: Ovum
Early cleavage o Cleavage and later developmental processes occur while the egg is still inside the uterus o Stage of Development: Zygote Diploid Large Chromosomes o Holoblastic – Entire cell dividing
EARLY DEVELOPMENT
development of an organism – increase in mass and elaboration of structures o cell activity – increase in cell number (due to mitosis), enlargement of cells, migration of cells, differentiation and specialization of cells
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
Classification of Eggs according to the relative amount of yolk which they contain: o microlecithal or oligolecithal – with little yolk Echinoderms, coelenterates, amphioxus & mammals except monotremes o medialecithal or mesolecithal – with moderate amount of yolk Eggs of annelid, mollusks, lampreys, lungfishes and ampbihibians 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 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
Prophase
Cleavage Metaphase
Anaphase
Telophase
Cleavage – not simply multiplication of cells but produces cells which, eventually, become arranged distinctly 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 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
Medialecithal egg (Frog) 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
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 surface of the embryo is prominent
©NexCab, JoFred, et. al (pictures). 2017
Medialecithal egg (Frog) o Animal pole – region where numerous smaller cells are found micromeres o Vegetal pole – region where fewer but bigger cells are found Macromeres
Microlecithal egg (starfish) o After the formation of a relatively large blastocoels o first sign: starfish embryo becomes flattened at the vegetal pole (the initial migration and differentiation of cells o cells will then invaginate within the blastocoel → formation of internal layers give rise to the endoderm and mesoderm germ layers 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 o Mesenchyme – large, stellate cells which are mesodermal in origin 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
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 animal pole The blastopore is now surrounded by dorsal, lateral and ventral lips o yolk plug – area of yolk that can only be seen within the confines of the blastopore; endodermal in origin o endoderm and chordamesoderm –from the expanding layer of cells which forms the roof of the archenteron o ectoderm – cells on the outer surface of the embryo o mesoderm – soon develops between the ectoderm and the endoderm. o Blastocoel – remnant of this may be seen. o Fertilization membrane – thin membrane that envelopes the gastrula
cut at animal pole only
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
Gastrula – 2 layered embryo from the ectodermal movements of cell
gastrocoel or archenteron (future primitive gut) - cavity created by the invagination of cells → digestive tract o blastopore – opening at the vegetal pole end; marks the posterior end of the embryo and becomes the future anus mouth of the in-pocketing of cells
The length of the gastrocoel indicates the specific stage of gastrula whether early, middle or late. Early gastrula exhibits a short gastrocoel whereas in late gastrula, the gastrocoel extends over half the length of the embryo and its apex may be seen a thin-walled vesicle
©NexCab, JoFred, et. al (pictures). 2017
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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 which they are accomplished is diffrerent from the medialecithal eggs. o Area opaca – the darker, outer, peripheral area seen in the specimens (due to the presence of yolk beneath it) o Area pellucida– the clearer, central area in the specimen. separated from the yolk by the segmentation cavity o Primitive streak – the thickened part of the blastoderm appears as a long streak in the center part of the specimen brought by the convergence of its surface layer toward the midline in the posterior half of the area pellucida. homologous to the epiboly in amphibians The cells that sink inside this thickened area forms a layer of cells between the epiblasts and the hypoblasts ectoderm – remaining cells on the surface after the immigration endoderm – innermost layer of cells near the blastocoel which has contributed some cells to the middle layer of sunken cells to form the mesoderm o Primitive groove – the central furrow o Primitive ridges – the thickened margins on both sides of the primitive groove o Primitive plate – thickening at the caudal end of the streak o Primitive pit – found at the cranial most end of the streak. This appears in the slide as a light area flanked by darkly-stained small streaks.
Hensen’s 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
NEURULATION
Neural Plate Stage – earliest stage o Neural Plate (aka medullary plate) – region of embryonic ectodermal cells (neuroectoderm), that lie directly above the notochord o Presumptive epidermis – remainder of the ectoderm after the formation of the medullary plate Forerunner of the outer layer of the integument o Notochord – oval mass directly beneath the neural plate and fr the mesoderm Posibbly fr the dorsal region of the archenteron roof Provides skeletal support during early development Temporary o Mesoderm – middle germ layer formed in the embryo during gastrulation Found on each side of the notochord and dorsal to the archenteron roof 3 regions of mesoderm in early organogenesis: Epimere – dorsal mesoderm; forms somites Mesomere – intermediate mesoderm; nephrotome; forms excretory system Hypomere – lateral plate mesoderm; delaminates into the inner splanchnic and outer somatic mesoderm o Endoderm – large yolk-ladden cells that surrounds the gastrocoel ©NexCab, JoFred, et. al (pictures). 2017
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
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
©NexCab, JoFred, et. al (pictures). 2017
