Embryogenesis and Fertility

Fertilization II

Recording Electrode and Clamping Electrode

• Clamping: reactive electrode set in a logical circuit that allows electrode to pass current in effort to keep measurement tin the recording electrode constant

• Maintains resting potential at -70 mV when sperm fuses despite change to +20mV upon fertilization

  • Leads to condition in which polyspermy can occur

  • Suggest that +20 mV prevents polyspermy

Mechanisms for Sperm Movement

Rheotaxis: Movement against bulk flow

• Facilitates upstream navigation of mammalian sperm cells

• Human sperm cells swimming in a spiral trajectory against bulk flow

• Fluid is moving from the site of the egg down the reproductive tract while sperm is moving against flow to find egg

Thermotaxis: Sperm can respond to thermal gradient

• Temperature surrounding oocyte is colder in comparison to other parts of the oviduct

• Sperm move toward colder region

Chemotaxis: Sperm can respond to gradients of molecules put out by oocyte and adjacent cells

• Final process that brings the sperm in proximity to the egg and its cells

• More complicated in mammals

Sperm Capacitation

• Changes that sperm undergo while in female reproductive tract to become fertilization-competent

Zona Pellucida

• Complex of glycoproteins

• Four zona pellucida proteins to create functional zona pellucida

• ZP2 cleavage model scientifically favored over ZP3 cleavage model

  • During fertilization, ZP2 plays dominant role in mediating the interaction between the sperm and zona pellucida

  • Upon exposure to released cortical granules, a disulfide bond is broken and a complex structural change of ZP2 occurs

  • Complex structural change in ZP2 prevents polyspermy

Izumo and Juno

• Izumo: ligand on the surface of the sperm

• Juno: receptor in the plasma membrane of the egg

• Juno is released in the form of vesicles to meet secondary sperm in the environment of the oocyte

  • Work to prevent polyspermy by interacting with sperm in environment surrounding oocyte 

• Izumo is a fusogenic protein that plays a role in fusing the sperm and oocyte

  • Cortical granules respond to fusion and release contents

Fertilization II

When the recording electrode enters the egg, records resting potential within the egg

• General cells have electrical properties, maintain some differential between the interior cell and exterior environment

• Resting potential: -70 mV

• Ion gates open and close during sea urchin fertilization

• When sperm approaches the egg, only one successfully fuses and there is an immediate change in the resting potential of the egg from -70 mV to +20 mV

  • Correlated: no other sperm can fuse, resting potential plays a part in blocking polyspermy?

  • Shifting clamped potential from +20 to -70 allows sperm to enter

  • Mechanism that is a fast-block to polysperm

• Speculation that the resting potential alters the configuration of molecules in cell membrane

• Requires energy to maintain, slow block is a chemical reaction with no energy expenditure

  • Works rapidly, gives slowblock chance to kick in, but inefficient energy-wise

• Primary model system in mammals: mouse

Mammalian fertilization

• Enfudibulum receives eggs from ovaries, immediately adjacent

• Mechanics of sperm to reach egg

  • Long path through biological material (tubular structures)

  • Chemotaxis doesn’t work well in environment, difficult to set up gradients/reliable cues

  • Most well established in vitro

• Rheotaxis 

  • Facilitates upstream navigation of mammalian sperm cells

  • Following a spiral trajectory along surface of tube, moving against bulk flow of material

  • Believed to drive motion of sperm in female reproductive tract

• Oviduct

  • When sperm enters the oviduct, changes sperm biologically and chemically (capacitation), stays for a while

  • Temperature gradient set up by temperature differential between the site of capacitation and where egg lies

  • Thermotaxis may play role in bringing sperm closer to egg

  • Sperm is capable of responding chemotaxically to various chemical agents emitted by egg, capable of undergoing chemotactic motion in respect to gradients likely established in the vicinity of the egg

• Most males deposit large amounts of sperm in female, but number of sperm that makes journey declines

• Egg has thick acellular membrane called the zona pellucida, composed of large glycoproteins that have complex sugar moieties attached to them

  • Mouse: 3 zona pellucida proteins (ZP1-3)

  • Often fourth in other mammals, mouse lost fourth due to mutation in the ZP4 gene (pseudo-gene)

  • ZP1 does not play an important role in mediating interaction with sperm

    • Sperm can still interact with egg although the frequency of fertilization drops

  • No formation of zona pellucida, can’t test the role of each protein

  • Most mammalian contain sperm acrosome

• Invitro experiments

  • ZP was purified and fractionated by standard biochem procedures

  • Effects on purified fractions on ability of sperm to fertilize egg was tested

  • Isolation of ZP, purifying individual components, test whether or not any of those components interfere with ability of sperm to interact in in vitro system 

  • Sperm goes under acrosomal reaction, others aren’t able to do so, effort to block polyspermy, mediate interaction between sperm and egg. Nature of molecular entities are different from sea urchin example

  • A fluid that sperm is migrating through is carrying purified material of ZP

  • Theory: The glycosylated site on ZP3 being the major initial interaction between sperm and ZP, argued that subsequently, in the release of cortical granules, that glycosylated site is destroyed from an enzyme of the cortical granules → explains blockage of polyspermy

• Shown that model couldn’t be case because you could genetically ensure that the glycosylated site didn’t exist, rendered unlikely via genetic manipulation

• If you lose ZP2 or ZP3, ZP cannot be made

• Make genetically hybrid mice in which genetic mutations could be rescued by addition of genes from humans → test role of various mouse ZP’s by guaranteeing the creation of a ZP through human proteins

• Initial interaction between sperm and ZP is due to ZP2, release cortical granules, disulfide bond that is broken and changes structure of ZP2, probably explains block to polyspermy

• Bindin directs interaction between sea urchin sperm and plasma membrane, may play role in fusion

• Receptor in plasma membrane of the egg called Juno

  • Interacts with ligand on surface of sperm named Izumo

  • Juno and one other component (maybe more) necessary for sperm binding and fusion of sperm with egg

  • Complex of molecules in plasma membrane of egg

  • Once egg is successfully fertilized, releases Juno components into its immediate environment, lost from surface of egg post-fertilization

→ block to polysperm

Early Embryogenesis I - Dr. Kankel

Embryogenesis

• Development starts with two pronuclei (one male and female) that fuse

• Using mice: ability to take oocyte and remove female pronucleus after meiosis to construct an oocyte w/ pronucleus of our choice

• Genomes of males and females are similar/almost identical, but two female and two male pronuclei lead to abnormal embryogenesis

  • Fails to develop due to genomic imprinting

Genetic Imprinting

• Epigenetic phenomenon

• Depending on the particular change that has occurred, alter the ability for a particular gene to be transcribed

• In genomic imprinting, only the allele form one parent will be transcribed

• Need both male and female pronuclei to make viable offspring

Early Stages of Embryogenesis

• Fertilization takes place toward the end of the oviduct

• Immediately after fertilization, divisions take place

• As embryo enters uterus, hatches from its extracellular membrane (zona pellucida)

• Hatched embryo attaches to the uterine wall and implants

Epididymis

• Head (caput)

• Corpus (body)

• Cauda (tail)

• The epididymis provides large quantities of relatively short RNA sequence to the sperm 

  • Produced in the epididymis, packaged into vesicles called epididymisomes, and fuse with developing sperm. RNa carried by sperm to oocyte

• Different parts of the epididymis produce different qualitiative spectrum of RNA products to the sperm

• Caput-derived embryos fail during early post-implantation

  • Cannot contribute to supportive development

Development

• Fusion of male and female pronuclei

• Cell division synchronous

  • 2-cell, 4-cell, 8-cell divisions

  • Developmental potential of cells t early stages are essentially equivalen

  • After 8 cell stage, 10 cell stage. No longer dividing synchronously

Compaction

• Cells begin to adhere to one another more itghtly

Cell divides into two lineages

Trophectoderm: will make no contribution to embryo-proper

Give rise to tissues of the placenta

Inner Cell Mass: population of cells contributing to embryo

Often referred to as embryonic stem cells

Capability of giving rise to all tissues an individual needs

Hatching of the Blastocyst

• Embryo eventually hatches from the zona pellucida

• Twins possible: aberrant hatching leads to twinning, each contains portion of inner mass

Implantation

• Occurs 7-10 days following fertilization

• Allows embryo to connect to the maternal vasculature

• Marks formal beginning of pregnancy

Gastrulation

• Upon implantation, inner cell mass undergoes gastrulation

• Transits from a ball of cells into layered structure → epiblast and hypoblast, undergo independent development

Results in the classical three-layered structure

• Ectoderm: outer layer

• Mesoderm: middle layer

• Endoderm: internal layer

• Done by a movement of cells originally located in the epiblast

Fate Map

• Where on the shield-like structure, what the cells in that location will give rise to in the developing embryo

• Draw fate maps at all different stages of development

• Cells that reside in a particular region in a developing embryo will result in a specific organ/manifestation later in development

Embryogenesis II - Dr. Kankel

Formation of Nervous System

• Early formation of the neural tube

• On the surface of the ectoderm

• Failure of tube closure, give rise to spina bifida

Spina Bifida

• Infant with an open spine

• Leave individual with loss of use of limbs, often legs

• LArger the opening, greater likelihood of death

• Can be medicated via folic acid

Spontaneous Abortion

• Meisois is relatively inefficient due to aneuploidies

• Frequency of aneuploidy rises significantly with maternal age 

• In some cases, spontaneous abortion can be attributed to aneuploidy

Teratologenesis - Dr. McAdow

Teratogens

• Exposure that has the potential to interfere with fetal development

• Chemical, medical envrionmental

• Drugs/medication, infections, radiation, hormones, maternal morbidities

• Major and or minor malformations

• Growth restriciton

• Miscarriage

• Placental abruption

• Stillbrith

• Long-term consequences not apparent at birth

Inductive Reasoning

• Exposure and degree of teratogenicity may be identified by the incidence of changes to organ systems

Cellular Responses

• Enzymes, substrates, genes (via mutation)

• Factors include: timing of exposure, dose, impact to fetus

• Fetus at greatest risk during organ differentiation

Pharmacology

Pharmacogenetics: Study of variability of drug response determined by single genes

Pharmacogenomics: Study of variability of drug response determined by multiple genes in the genome

Pharmacokinetics: Pertains to the degree in which genetic variability can impact absorption, bioavailability, distribution, metabolism, and excretion of drugs

Pharmacodynamics: Pertains to the degree in which genetic variability can impact biochemical, physiologic, and molecular effects of drugs

Examples

Congenital Rubella: RNA virus transmitted through respiratory droplets, results in congenital rubella

Timing of exposure key

Congenital Cytomegalovirus (CMV): May impact sensorineural hearing, vision, intellectual ability

Dose of exposure is key

Neural Tube Defects: Results from medication exposure (anti-epileptics, accutane), maternal hyperglycemia, folate deficiency

Timing of exposure key

Fetal Function and Physiology I - Dr. Stiller

Goals of Maternal Physiology

• Fetus and mother must survive pregnancy 

• Fetus and mother must survive delivery

Evolution of Pregnancy

• Development of antibiotics, bloodbanks, advancements in anesthesia, pregnancy testing capabilitie, medical images, ultrasounds

• Decreased maternal mortality

Uterus

• The released egg, ova, is scooped up by the fimbrae of the fallopian tube following ovulation

• Ova is fertilzied in the fallopian tube 

  • Ectopic pregnancy: when the fertilized ova implants itself into the fallopian tube 

• Fertilized ova travels to the uterus for implantation

  • The vasculature of the uterus provides a safe environment for the developing fetus

  • The uterus can also grow to accomodate the growing fetus

The Placenta

• Area of gas, nutrition, and waste exchange

• Produces hormones → progesterone in early pregnancy

• Two umbilical arteries and one umbilical vein

  • Umbilical arteries contain deoxygenated blood form the fetus and flows to the placenta → maternal circulation

  • Umbilical vein contains oxygenated blood from the placenta and flows to the fetus

Maternal Cardiovascular System Preparation

• The mother experiences an increase in clotting factors prior to childbirth to prevent excess blood loss

  • Increase in clotting factors increased likelihood of deep vein thrombosis

• Muscle contraction of the uterus compresses blood vessels, preventing hemorrhage

• The increase in blood volume prior to childbirth prevents mother from going into hemorrhagic shock

Maternal Osmoregulation

• Osmolality in the mother decreases to help accommodate for water transportation to the developing fetus

  • Normal osmolality = 290 mOsm/kg

  • During pregnancy = 275-280 mOsm/kg

• Changes to maternal water and salt metabolism

Hemodynamic Changes

• Blood volume mL +40%

• Systemic vascular resistance -21%

Maternal Respiratory Function Changes

• Non-pregnant → Tidal volume: 450        Residual volume: 1000

• Pregnant → Tidal volume: 650                Residual volume: 800

• Hyperventiation of person who is pregnant

  • Lowers the carbon dioxide in the blood

  • Increases pH to facilitate the Double Bohr effect

  • Increases uterine blood flow

  • ⅙ of cardiac output is dedicated to the uterus

• Tidal volume: volume exchanged with the tide of our breath at rest

  • During pregnancy, goes up 40%

• Residual volume: air in lungs not expires with each breath

Double Bohr Effect

• Oxygen affinity increases are carbon dioxide levels decrease, resulting in an inceease in pH, as fetal blood travels through the placenta

• As pH changes in the blood, oxygen affinity changes

• Unloading CO2 to the amternal compartment, CO2 level changes enough that it changes pH, changes hemoglobin affinity for oxygen

• Maternal hemoglobin has decreased affinity for oxygen and fetal hemoglobin has increased affinity for oxygen (greater o2 saturation)

Fetal Function and Physiology II

Fetus

• Steals substrates from the mother due to low capacity for glucogenesis

• Alterations to the carbohydrate metabolism allows for a continuous supply of glucose to the fetus

• Alterations in glucose and insulin levels can result in gestastional diabetes and fetal macrosomia

  • Excessive fetal growth

Glucose Control

• An adult sotres glucose in liver as complex carbs

• Fetus has low capacity for glucogenesis due to low arterial PO2, dependent on maternal glucose being higher than fetal glucose

• Human somatomammotropin: hormone made by placenta to create insulin insensitivity to promcote hyperglycemia and hyperinsulinemia

Fetal Respiratory System

• The fetus practices muscular breathing throughout their gestational development

• Surfactant is important to help reduce surface tension and prevents alveoli from collapsing

  • At 35/36 weeks of gestation, surfactant produced

  • Premature infants may develop respiratory distress syndrome (SDS) due to absence of surfactant at birth

Saccular and Alveolar Development

• 16 Weeks: Development of terminal bronchioles

• 19 Weeks: Development of respiratory bronchioles

• 28 Weeks: Development of transitional duct and saccules (site of air exchange)

Amniotic Fluid

• Cushion for fetus (allow muscles to develop, stretch)

• Being swallowed to help create canals of GI system

• Breathed in and out to help practice breathing and develop lungs

Abnormalities

Polyhydramnios: too much fluid (diabetes, esophageal atresia, anencephaly)

Oligo/Anhydramnios: too little or no fluid at all (IUGR, renal agenesis)

Maternal Antibody Transfer

• Placenta is bridge between mother and growing fetus for food, water, antibodies

• Maternal IgG antibodies can cross placenta via endocytosis, providing infant with passive immunity for first few months of life until inoculations can be obtained

  • Further passive immunity can be provided/strengthend through breastfeeding

• Diseases can be transferred

  • Grave’s, Maternal Rhesus Isoimmunization, Maternal Myasthenia Gravis, Maternal Immune Thrombocytopenia

Cardiovascular System

• 8 Weeks: heart is partially developed

  • Chambers not fully developed

• Fetal cardiovascular systems consists of two systems working in parallel with one another, adult system is a series circuit

• Fetal circualtion: Left heart system and right system work in parall

Ductus Arteriosis: Allows blood from R ventricle to bypass pulmonary ciruclation to the aorta

Foramen Ovale: Allows blood from R attrium to bypass pulmonary circulation to the L atrium

Biventricular Cardiac Output

• R ventricle is responsible for ⅔ of cardiac output, mostly distributed to the body and placenta

• L ventricle responsible for distributing blood to head, heart

• Both R and L ventricles pump blood into systemic system

Transition from Fetal to Newborn Circulation

• Once the umbilical cord is clamped, there is no further blood flow from the ductus venosus

  • Discontinues placental circulation

  • Newborn systemic vascular resistance increases, blood flow directly to fetal lungs

  • When increased blood returns to the pulmonary vein occurs, increase in pressure to the left atrium, resulting in closing of foramen ovale\

Assessing Fetal Growth via Ultrasound

• Approximately fetal weight is generated using head circumference, biparietal diameter, abdominal circumference, and femur length

• Liver weight can indicate gestational age

Intrautierine Growth Restriction

• Usually defined as weight that is less than the 10th percentile, implying pathological growth pattern

Symmetric: All measurements line up, early insult

Assymetric: All the measurements do NOT line up

• Head larger than body

• Brain sparing: fetus has adaptations when under stress to devote more nutrients to brain development

• Later insult

Fetal Doppler to Assess the Placenta

• Uses doppler effect of red blood cells moving through the blood vessels that bounce back at a certian frequency to measure blood flow through various vessels in the placenta and fetus

Middle Cerebral Artery Doppler

• Located in middle of brain in Circle of Willis

• Measures velocity to assess brain sparing and fetal anemia

• Higher velocity is indicative of brain sparing

• Heart pumps faster in fetuses with fetal anemia

Umbilical Artery Doppler

• Measuring the ratio of systolic peak to the diastolic low

Ductus Venosus Doppler

• Indicates function of right side of the heart