W6 - FERTILISATION AND EMBRYO DEVELOPMENT

Question 1

What are the two phases of the development of the foetus? Explain embryology

Answer 1

• The development of the foetus is divided into two phases
  
  • Prenatal
    
    • Before birth
  • Postnatal
    
    • After birth
• Embryology is the study of embryos and foetuses
  
  • Prenatal development
    
    • Embryonic period - fertilisation until the end of the 8th week
      
      • Placenta develops
      • All major adult organs are formed (gastrulation and organogenesis)
    • Foetal period - 9th week until birth
      
      • Growth and development

Question 2

How long is an oocyte viable for? What is the duration of pregnancy?

Answer 2

• An oocyte is viable for 12-24 hours after ovulation
  
  • Most sperm retain fertilisation power for 24-48 hours after ejaculation
  • Thus, coitus must occur no more than 2 days before ovulation and not later than 24 hours after for fertilisation to occur
  • Need to allow 4-6 hours for sperm transport and capacitation
• At the optimal time, the oocyte is about 1/3 of the way down the length of the uterine tube
  
  • Ovum retained in the ampulla of the uterine tube - allows time for uterine lining to be adequately prepared by progesterone from the luteal phase of the ovarian cycle
• Pregnancy will take approximately 38 weeks (266 days) form zygote to birth - spread over three trimesters
  
  • The due date for birth is calculated from 4 weeks from the first day of the last normal menstrual period (LNMP)
  • 40 weeks (280 days or 9 months plus 7 days)

Question 3

What is the clinical significance of embryology?

Answer 3

• Clinical significance
  
  • Pre-implantation developmental abnormalities
    
    • Failure to implant within the uterus - failed or ectopic pregnancy
    • Reason for most problems in pregnancy
  • Severe embryonic period abnormalities
    
    • Teratogen exposure and infection during week 1-8 - neural tube and other defects
      
      • Often quite severe - disrupts organ system development
  • Foetal period developmental abnormalities
    
    • Malformation or mechanical abnormalities - talipes
• Important to know the developmental point of the embryo/foetus so that the effects of the trauma can be more accurately identified

Question 4

What are some reasons why sperm may not reach the oocyte?

Answer 4

• Main reasons
  
  • Fall out of vagina
  • Trapped in crypts, glands and mucus
  • Run out of energy
  • Destroyed by environment (Acidic nature of the vagina)
  • Incapable of fertilisation due to abnormal shape/reduced motility
  • Do not pass down the correct fallopian tube
• Only a few thousand make it into the uterine tube

Question 5

Explain the week 1 events of embryo development

Answer 5

• Fertilisation
• Cleavage and blastocyst formation
• Blastocyst begins to implant in endometrium

Question 6

Explain the process of sperm capacitation

Answer 6

• Freshly ejaculated sperm cannot immediately fertilise the oocyte
• They need to spend several hours in the female reproductive tract to attain a capacity to fertilise
  
  • Their motility must be enhanced through the cervical mucus, uterus and uterine tubes
• Oestrogen and vaginal mucus destabilise the sperm plasma membrane and trigger hyperactive motility
• The process involves
  
  • Removal of the protein coating acquired in the epididymis
  • Reorganisation of plasma membrane to expose binding sites
• This prevents the spilling of acrosomal enzymes
  
  • Fragile acrosomal membranes could rupture prematurely in the male reproductive tract, causing some degree of autolysis of male reproductive organs
• The sperm appear to “sniff” their way to the oocyte - olfactory receptors that respond to chemical stimuli
  
  • Oocyte and surrounding cells release signalling molecules that direct the sperm
• Capacitation - secretions from the female tract and oestrogens weaken the sperm’s protective protein coat acquired in the epididymis
• Reorganisation of plasma membrane to expose binding sites and hypermotility is triggered

Question 7

Explain the acrosome reaction

Answer 7

• Swelling of the acrosome and fusion of the outer acrosomal membrane with overlying plasma membrane
• Calcium-dependent event
  
  • Increase intracellular Ca2+ and cAMP
• Stimulated by progesterone and a protein (ZP3) on the zona pellucida that surrounds the oocyte
• Release of digestive enzymes from the acrosome
  
  • Hyaluronidase - cumulus cell penetration
  • Acrosin - zona pellucida digestion
• Acrosome-reacted sperm have a very short lifespan
• Process
  
  • Once in the vicinity of the oocyte, sperm weaves its way through the cells of the corona radiata, assisted by cell-surface hyaluronidase on the sperm - digests the cement between the granulosa cells in the area
  • After breaching the corona, the sperm head binds to ZP3 of the zona pellucida (functions as a sperm receptor)
    
    • The binding, and progesterone, opens Ca2+ channels, leading to a rise in Ca2+ inside the sperm which triggers the acrosomal reaction
  • Acrosomal reaction - release of acrosomal enzymes that digests holes in the zona pellucida
    
    • Single sperm can fertilise the egg and penetrate the zona pellucida
  • Once the path is cleared, the sperms tail gyrates, forcing the sperm’s head to move towards the oocyte membrane
  • Sperm’s post-acrosomal “collar” binds the oocyte’s plasma membrane receptors causing
    
    • Oocyte to form microvilli that surround the sperm head, and the sperm and oocyte membranes fuse
    • Cytoplasmic contents of the sperm enter the oocyte, leaving the sperm’s plasma membrane behind
      
      • Gametes fuse together with perfect contact, causing the contents of both cells to combine within a single membrane - all without spilling

Question 8

Explain the process of fertilisation

Answer 8

• Capacitation
  
  • Oestrogen/mucus destabilise sperm plasma membrane and cause hyperactive motility
• Acrosome reaction
  
  • Progesterone and binding to ZP3 causes increase Ca2+ and release of digestive enzymes from sperm
• Fertilisation
  
  • Sperm digests zona, binds to sperm-binding receptor and fuses with oocyte plasma membrane
• Polyspermy block
  
  • Fusion causes increase Ca2+ and electrical block of oocyte plasma membrane (fast block), then cortical granule release which hardens the zona and removes sperm-binding receptors (slow block)

Question 9

Explain the polyspermy block

Answer 9

• Once sperm has entered the oocyte, Ca2+ is released in waves by the endoplasmic reticulum into the cytoplasm
  
  • Activated oocyte to prepare for 2^nd meiotic division
  • Cortical reaction - granules located just inside the plasma membrane spill their enzymes into the extracellular space beneath the zona pellucida - zonal inhibiting proteins
    
    • Destroys the sperm receptors, preventing any more sperm from entering
    • Spilled material binds to water, and as the material swells and hardens, it detaches all sperm still bound to the receptors on the oocyte membrane, completing the slow block
• Electrical block - attachment of a sperm to ovum surface induces membrane depolarisation and increased permeability to Ca2+, triggering an electrical block on the surface of the ovum, preventing the fusion of other sperm

Question 10

Explain the completion of meiosis II and fertilisation

Answer 10

• Sperm loses its plasma membrane as the cytoplasmic contents of the sperm enter the oocyte
  
  • The centrosome elaborates microtubules from its midpiece, which are used by the sperm to locomote its DNA-rich nucleus toward the oocyte nucleus
  • During this, the nucleus swells to about 5 times the normal size to form the male pronucleus
  • The secondary oocyte (activated from its semidormant state by the Ca2+ influx) completes meiosis II, forming the ovum nucleus and 2^nd polar body
  • Accomplished ovum nucleus swells, becoming the female pronucleus and the two pronuclei approach each other
  • Syngamy - the two pronuclei come together to form the zygote
  • As the mitotic spindle develops between them, the pronuclei membranes rupture, releasing their chromosomes together into the immediate vicinity of the newly formed spindle
  • Almost as soon as the male and female pronuclei come together, their chromosomes replicate
• Zygote is now ready to undergo the first mitotic division of the conceptus

Question 11

Explain the pre-implantation development process

Answer 11

• Zygote - the fertilised egg
• 4-cell stage - day 2
• Morula - solid ball of blastomeres - day 3
• Early blastocyst - morula hollows out, fills with fluid and “hatches” from the zona pellucida - day 4-5
• Implanting blastocyst - consists of a sphere of trophoblast cells and an eccentric cell cluster called the inner cell mass - day 7

Question 12

What are the stages of pre-implantation?

Answer 12

• Cleavage stage - 2-8 cells
  
  • Totipotent blastomeres (has not undergone differentiation; generate complete individual including placenta)
  • Period of fairly rapid mitotic divisions of the zygote without intervening growth (increase in cell number without increase in cell size)
  • Goal - to produce small cells with a high surface-to-volume ratio - enhances their uptake of nutrients and oxygen and disposal of wastes
  • Provides a large number of cells to serve as building blocks for constructing the embryo
  • About 36 hours after fertilisation - the first cleavage division of the zygote produces two identical daughter cells - blastomeres
• Morula stage - 16-32 cells
  
  • Inner and outer cell populations formed (beginning of differentiation)
    
    • Determines the future fate of the cells
    • Gap junctions between inner cells
    • Tight junctions between outer cells
  • By 72 hours, a loose collection of cells that form a berry-shaped cluster of 16-32 cells called the morula has been formed
  • Transport of the embryo continues towards the uterus
  • Na+ pumped into the morula - influx of water
• Blastocyst stage - >64 cells
  
  • First cell differentiation event (trophectoderm and inner cell mass formation)
  • Pluripotent inner cell mass (generate complete individual excluding placenta)
  • Accumulating fluid, floating free in the uterus
  • Rapid growth and enzymes - hatching from the zona pellucida - zona pellucida starts to crack due to the swelling blastocyst, blastocyst “hatches” from it
  • Blastocyst now a fluid-filled hollow space composed of a single layer of large, flattened cells called trophoblast cells and a small cluster of 20-30 rounded cells called the inner cell mass
  • Increase in CDX2 in outer cells - trophectoderm differentiation
  • Increase in OCT4 in inner cells - inner cell mass differentiation

Question 13

Explain the week 2 events of embryo development

Answer 13

• Implantation completed
• Placenta and extraembryonic membranes begin development
• Development of inner cell mass proceeds

Question 14

Explain the process of implantation

Answer 14

• Adherence (day 6)
  
  • Protein-mediated binding of trophoblasts to endometrium
    
    • Outer trophoblasts mediate the process
  • Digestive enzymes from trophoblasts erode the endometrial layer - it embeds itself
    
    • Implants high on the uterine wall - if the endometrium is not optimally mature, the blastocyst detaches and floats to a lower level with proper receptors and chemical signals
      
      • Higher = better (thick endometrium and myometrium)
    • Secretion of hormones and proteolytic enzymes from blastocyst causing the neighbouring endometrial cells to degenerate
• Endometrium quickly thickens at the point of contact and takes on characteristics of acute inflammatory response
• Invasion (day 7)
  
  • Trophoblast grows to form cytotrophoblast and syncytiotrophoblast
    
    • Cytotrophoblast - cell in inner layer retain their cell boundaries
    • Syncytiotrophoblast - loose plasma membranes and form multinuclear cytoplasmic mass - these cells digest and invade the endometrium
  • Human chorionic gonadotropin (hCG) is produced for the first time
• Completion (day 12)
  
  • Blastocyst enveloped by endometrium
    
    • Endometrium is eroded so the blastocyst can burrow in
    • Endometrial cells proliferate to cover and seal the implanted blastocyst
  • Syncytiotrophoblast secretes hCG - directly stimulates the corpus luteum to produce progesterone (maintains the pregnancy for 8-12 weeks)
    
    • Maintains secretion of oestrogens and progesterone
    • Oestrogen stimulates prostaglandins from the endometrium of the uterus - increases vascular permeability and swelling of the endometrium

Question 15

What is the bilaminar embryonic disc and extraembryonic membranes?

Answer 15

• Inner cell mass differentiates into an epiblast and hypoblast
• Fluid-filled cavities form in epiblast (amnion) and hypoblast (yolk sac) - bilaminar embryonic disc (contains epiblast on the top and hypoblast on the bottom)
• Out-pocket hypoblast cells form allantois (early base of the umbilical cord)
  
  • Beginning of the placenta
• Extra-embryonic mesoderm, cytotrophoblasts and syncytiotrophoblasts form chorion and chorionic villi - beginning of foetal placenta

Question 16

Explain the function of extraembryonic membranes

Answer 16

• Amnion
  
  • Prevents physical trauma, maintains temperature, avoids embryonic structure fusing, permits movement
• Yolk sac
  
  • Brief nutrient support, part of future gut, source of early blood cells and vessels
• Allantois
  
  • Forms base of umbilical cord
• Chorion and chorionic villi
  
  • With maternal decidua forms true placenta

Question 17

Explain the week 3 events of embryo development

Answer 17

• Appearance of the primitive streak
• Gastrulation - formation of three primary germ layers

Question 18

What is the primitive streak?

Answer 18

• Midline groove (primitive streak) forms along caudal end of bilaminar embryonic disc
• Primitive streak establishes longitudinal body axis (head and tail) of embryo
• Epiblast cells migrate medially across the other cells and enter the primitive streak
  
  • First cells to enter the groove displace the hypoblast cells and form the more inferior layer (endoderm)
  • The next ones push laterally between the cells at the upper and lower surfaces (mesoderm)
  • Once the mesoderm is formed, the mesodermal cells immediately beneath the streak aggregate - form a rod of mesodermal cells called the notochord (first axial support of embryo)
  • Remaining cells on the dorsal surface form the ectoderm

Question 19

Explain the process of gastrulation

Answer 19

• Two layered embryonic disc transforms into a three-layered embryo in which the primary germ layers are present
  
  • Ectoderm
    
    • Remaining epiblast
  • Mesoderm
    
    • Fill the middle-layer of the embryo
  • Endoderm
    
    • Displace cells of hypoblast

Question 20

Explain the week 4-8 events of embryo development

Answer 20

• Differentiation of ectoderm, mesoderm and endoderm
• Organogenesis - formation of organ system

Question 21

Describe the differentiation of the three primary germ layers

Answer 21

• All adult tissues are derived from the ectoderm, mesoderm and endoderm
• Each layer has a pre-established “fate”
  
  • Ectoderm
    
    • Nervous system, skin epidermis
  • Mesoderm
    
    • Most blood vessels, connective tissues, bone and muscle
  • Endoderm
    
    • Epithelial linings and associated glands of the digestive, respiratory and urogenital systems

Question 22

What are the ectoderm derivatives?

Answer 22

• Forms
  
  • Epithelium - skin, hair, nails, sensory epithelium, pituitary gland
  • Connective tissue - bones and blood vessels of the head
  • Nervous system - brain, spinal cord, peripheral nervous system
• Neurulation - differentiation of ectoderm that produces the brain and spinal cord
  
  • Induced by chemical signals from the notochord
    
    • Ectoderm overlying the notochord thickens forming the neural plate
    • Ectoderm starts to fold inward as a neural groove, forming neural folds as it deepens
  • By day 22, the superior margins of the neural folds fuse, forming a neural tube which pinches off from the ectodermal layer and becomes covered by surface ectoderm
• By the end of 4 weeks, the three primary brain vesicles (prosencephalon, mesencephalon, and rhombencephalon) are present

Question 23

What are the mesoderm derivatives?

Answer 23

• Forms
  
  • Epithelium - serous membranes, kidney, gonads, ducts
  • Connective tissue - bone, cartilage, connective tissue proper
  • Muscle - cardiac, skeletal, smooth
• Differentiation
  
  • First evidence is the appearance of the notochord
    
    • Eventually replaced by vertebral column
    • Three mesodermal aggregates appear on either side of the notochord - somites
      
      • On the side of these are small clusters of segmented “intermediate” mesoderm, then double sheets of lateral plate mesoderm
• Somites
  
  • Sclerotome - cells migrate medially and gather around the notochord and neural tube, producing the vertebra and rib at each level
  • Dermatome - cells help for the dermis of the skin in the dorsal part of the body
  • Myotome - cells develop in conjunction with the vertebrae to form the skeletal muscle of the neck, body trunk and limb buds (later muscles of the limbs)
• Intermediate mesoderm - gonads and kidneys
• Lateral plate mesoderm
  
  • Paired mesodermal plates - somatic mesoderm (skin dermis, parietal serosa lining the ventral body cavity and most tissue of the limbs) and more inferior splanchnic mesoderm
    
    • Provides mesenchymal cells that form the heart and blood vessels and most connective tissue of the body (almost the entire wall of the digestive and respiratory organs)
  • Cooperate to form the serosae of the coelom or ventral body cavity

Question 24

What are the endoderm derivatives?

Answer 24

• Forms
  
  • Epithelium - lungs, gastro-intestinal lining, gall bladder, pancreas, urinary tract lining
  • Organs of the gastrointestinal tract (pharynx, oesophagus, etc)

Question 25

Explain the process of foetal development

Answer 25

• Foetal period (week 9 to birth)
• Rapid growth and weight gain after this time
  
  • From 22mm (2g) to 55mm (3.2kg)
• Bones begin to ossify, and skeletal muscles are well formed and contracting spontaneously (movements felt by mother - 5^th month)
• Further differentiation and refinement of body structures is required
• Blood delivery to and from the placenta via the umbilical vessel is constant and efficient
• Heart and liver are competing for space and form the conspicuous bulge on the ventral surface of the embryo’s body
• Survival if born prematurely after 27-28 weeks