Regulation of early embryo development Flashcards
Aims and objectives
Aims
- To explain the regulation of early embryo development, to integrate environmental (maternal) and embryonic regulatory mechanisms
Objectives
- Describe regulatory mechanisms involved in the control of early embryo development
- Consider the integrative relationship between the early embryo and its environment to result in normal development
- Develop a critical argument relating environmental impact during early embryo development to potential long-term consequences by selecting relevant evidence from the literature
What is the periceonceptional period?
The periconception period, defined as the 14 weeks before and 10 weeks after conception, is a critical window with a substantial impact on fetal growth and development. Within this period, gametogenesis, organogenesis and placental development occur.
This is the time of oocyte and sperm development
Environmental sensitivity in early development can lead to syndomes such as ‘Large offspring syndrome’ can you explain what this is?
How does this relate/ translate to humans?
Large offspring syndrome in cattle and sheep
Large offspring syndrome: a bovine model for the human loss-of-imprinting overgrowth syndrome Beckwith-Wiedemann
In mammals’ fertilization occurs in the fallopian tube which has unique conditions which are unknown as not much research into this area
Major epigenetic reprogramming takes place that is crucial for the normal fate of the embryo.
Very vulnerable to changes in environmental conditions such as the ones implied in IVF (IVF tries to replicate that environment seen in the fallopian tubes)
Including in vitro culture, nutrition, light, temperature, oxygen tension, embryo-maternal signalling, and the general absence of protection against foreign elements that could affect the stability of this process.
There has been a change in phenotype observed for example in large offspring syndrome (LOS).
It is characterized by large size at birth, gross abnormalities in different organs, mainly visceromegaly (enla, and metabolic alterations, especially in the glucose-insulin system, hypoglycaemia, large tongue and umbilical hernia.
All these features are like those found in the Beckwith Wiedemann syndrome in humans
What is the Dutch Winter Famine and what did this help us understand?
What effects did it have on the offs[ring who were exposed to this?
The Dutch famine was when a German Nazis blockade cut off food and fuel shipments from farms and towns
This led to hunger in the womb
High incidence of heart disease, impaired glucose tolerance and higher BMI if exposed to famine
Another excellent example the 1944/45 dutch famine which provides us with that ability to look at a large cohort.
Exposure to the famine at mid or late gestation led to impaired glucose tolerance
But exposure to the famine in early gestation led to atherogenic lipid profile, higher BMI and increased risk of congenital heart disease
What are the main stages that happen during the periconceptional period?
- Oocyte maturation
- Fertilisation
- Acrosome reactions
What happens during the process of oocyte maturation during the periconceptional period?
Oocyte maturation:
- oocytes are held in meiotic arrest in prophase I prior to ovulation.
- LH (luteinising hormone) surge promotes the resumption of meiosis of the arrested oocytes
- Germinal vesicles breakdown and their progression through the second meiotic cycle,
- Arrest again at metaphase II until fertilization.
What happens during the process of Fertilisation during the periconceptional period?
Fertilisation:
- Capacitation
- Male takes 3 months to produce mature sperm
- spermatozoa must undergo capacitation, which allows them to bind to and penetrate the oocyte
- There is removal of the seminal proteins from the surface of the spermatozoon, alteration of glycoproteins on the sperm plasma membrane and an efflux of cholesterol resulting in an increase in the membrane fluidity.
What happens during the Acrosome reactions during the periconceptional period?
Acrosome reaction:
- the acrosome reaction can occur.
- The acrosome is produced in the Golgi system; several small proacrosomal vesicles are produced by fusion with several smaller vesicles, one grows and migrates towards the nucleus forming the acrosome.
- Attachment to the zona pellucida results in activation of calcium channels and a release of intracellular calcium, which activates cAMP and phosphokinase A pathways.
- The acrosomal membrane then fuses with the sperm head and releases lysins which lyses the zona pellucida; the cell membrane of the spermatozoon and the oocyte are then able to fuse.
- Once sperm penetration has occurred, the zona pellucida undergoes modification to change it into a protective outer layer for the developing embryo.
- Once this fusion has occurred, the spermatozoon’s tail stops beating immediately, and the sperm is drawn into the oocyte by elongation. A massive influx of sodium ions enters the oocyte causing depolarisation, this alteration prevents polyspermy
Oocyte maturation and fertilisation
What happens/ the processes that the human embryo goes through during the periconceptional period?
- Finally, meiosis resumes and there is extrusion of the second polar body.
- Following this process, the two pronuclei appear; these contain the genetic material from the spermatozoa and the oocyte. They migrate towards one another and following the disappearance of their membranes, the chromosomes within them combine to form the single zygote nucleus.
- The zygote then undergoes mitotic divisions to two cells, four cells and eight cells at approximately 27.9, 40.7- and 59.1-hours post fertilisation.
- This mitosis does not contain growth stages like cell division in adult cells but simply a synthesis or S phase (during which DNA replication occurs), followed by mitosis (the separation of the chromosomes) and then cytokinesis (separation of the cell components). This means that the resulting cells (or blastomeres) have a reduced cytoplasmic volume with each cell division.
- Once the embryo reaches the eight or sixteen cell stage, it starts to compact within the zona pellucida: tight junctions form between the external blastomeres resulting in a sealed sphere.
- Between the internal blastomeres, gap junctions form allowing the movement of molecules and ions between the cells.
- At approximately 86.6 hours post-fertilisation, the embryo contains in the region of 30 cells and is termed a morula.
- The external cells will give rise to the trophectoderm (forms placenta) and the internal cells to the inner cell mass (forms baby) once the blastocyst is formed.
- The outer blastomeres start to express sodium transporters; the movement of sodium then creates an osmotic effect and causes a blastocoel cavity to form.
- By day 5 (120 hours post-fertilisation, on average 104.1 hours) the human embryo should be at the blastocyst stage of development.
- A human blastocyst consists of between 80 and 160 cells and comprises of an inner cell mass (ICM) (approximately 30% of cells) and a trophectoderm (TE).
- Finally, the blastocyst starts to hatch out of the zona pellucida.
What are the stages that occur in the early and late embryo in embryo metabolism?
Early embryo
- Low metabolic activity
- Low capacity to regulate intracellular homeostasis (Ph, ion content)
- High susceptibility to environmental disturbance
Late embryo
- Increased metabolic activity
- Better capacity to regulate intracellular homeostasis (Ph, ion content)
- ?? Reduced susceptibility to environmental disturbance
What can embryo metabolism be altered by?
How does IVF effect this?
Embryo metabolism is known to be altered by several environmental factors including oxygen tension (Wale and Gardner, 2012) and whether cultured in groups or individually.
Talk about PROMOTE trial
IVF requires incubation of the embryo and therefore a standard incubator is used then this can effect the conditions in which metabolism is occuring in:
Hence, fluctuations in temperature, oxygen and CO2caused by the requirement to remove embryos from standard benchtop incubators for observation may also induce metabolic changes in the embryo.
What are the different incubators which can be used for amino acid metabolism?
study looks at the two different types of incubators
Time lapse takes photo within incubators so don’t have to disturb their environment and the standard is where they must be taken in and out to look at
Tell me about gene expression in embryos
Translation into protein is continued throughout the preimplantation period.
Messager (mRNAs) inherited from the oocyte (maternally inherited) regulate embryo development early on.
During early cleavage, the embryonic genome is gradually switched on (MZT: maternal-zygotic transition or ZGA: zygotic genome activation) to initiate de novo transcription.
What are some environments that could influence in embryos?
Follicular fluid
Fallopian tube
Uterus
How can the follicular fluid influence?
Human follicular fluid composition is influenced by maternal BMI, PCOS and/or insulin resistance.
Lipid or amino acid profiling reveal relationships to maternal status.
For example, maternal obesity is linked with elevated TG and insulin in serum and in follicular fluid.
Specific FA (mainly NEFA) are increased in follicles from obese women.
Human follicular fluid composition is influenced by maternal BMI, PCOS and/or insulin resistance.
Lipid or amino acid profiling reveal relationships to maternal status.
For example, maternal obesity is linked with elevated TG and insulin in serum and in follicular fluid.
Specific FA (mainly NEFA) are increased in follicles from obese women.
How does the uterine fluid influence?
Uterine fluid composition is distinct from serum in the pregnant mouse
A similar distinction is found in non-pregnant humans
study looked at uterine fluid in those non-pregnant and whether they had a healthy or non-healthy diet
Showed different in AA between these diets
How do these environments influence and the effects it leads to?
LPD increases weight, blood pressure, increased activity and reduced memory
What does a maternal LPD affect?
Uterine fluid
How does maternal low protein diet effect blastocysts and cell lineages?
Blastocyst lineages
- more cells
- specifically more Trophoectoderms
Blastocyst invasiveness
- Increased spreading in vitro
What is mTORC signalling and what is its mechanism of action?
mTORC= mammalian target of rapamycin
and this regulates cell proliferation, autophagy by participating in multiple signalling pathways
How does maternal obesity in humans effect blastocyst lineages and metabolism?
How can we model the effects of IVF? What animals do we use and why?
Use rodents because…
- they have short intergenerational periods
- Possess a haemochorial placenta, more like that of humans than the epitheliochorial placenta of domestic species
How can the in vitro cultured-produced males affect the following generations?
In vitro cultured-produced males had lower sperm concentrations (5.8 × 106 spermatozoa in IVC vs. 14.5 × 106 spermatozoa in control), and these sperm exhibited decreased overall motility (49.6% vs. 72.8% in control) and progressive motility (22.6% vs. 32.2% in control).
Fertility tests demonstrated that the percentage of pregnancies was reduced for IVC males (35% for IVC-produced males vs. 86% for in vivo controls).
we analysed, through gene silencing, the effect of IVC on the mRNA expression at the blastocyst stage for 11 known gene expression modifiers of epigenetic reprogramming. Suboptimal IVC reduced the expression of Kap1, Sox2, Hdac1, Dnmt1, and Dnmt3a, suggesting a molecular epigenetic role for gene expression modifiers in the origin and transmission of these abnormal phenotypes.
Glucose intolerance was shown only in F1 and F2 male.
The same occurred with male abnormalities in the organ size of the liver, which were transmitted to F1 and F2 males
Methylation of CpG islands in regulatory regions can influence expression of what?
Methylation of CpG islands in regulatory regions (eg promotors) can influence expression of specific genes (mainly repression). regions of DNA where a cytosine nucleotide is followed by a guanine nucleotide in the linear sequence of bases along its 5’ → 3’ direction.
Tell me about the early embryo and DNA methylation
Methylation marks are erased in PGCs.
Oocyte and sperm continue to re-acquire methylation marks until/during maturation yet in different time frames and to different extents.
During preimplantation development, demethylation of the genome occurs before re-methylation begins at the blastocyst stage in a cell- type specific manner (ICM vs TE).
The first wave occurs in the germline, initiated with the erasure of global methylation in primordial germ cells (PGCs) and completed with the establishment of sex-specific methylation patterns during later stages of germ cell development.
The second wave occurs after fertilization, including the erasure of most methylation marks inherited from the gametes and the subsequent establishment of the embryonic methylation pattern. The two waves of DNA methylation reprogramming involve both distinct and shared mechanisms.
Why is methylation so susceptible?
What are the mechanisms to distinguish between?
A widely accepted hypothesis for the evolution of genomic imprinting is the ‘parental conflict hypotheses” aka the “kinship theory”. What does this hypothesis state?
What supports this hypothesis?
This hypothesis states that the inequality between parental genomes due to imprinting is a result of the differing interests of each parent in terms of the evolutionary fitness of their genes.
The father’s genes that encode for imprinting gain greater fitness through the success of the offspring, at the expense of the mother. The mother’s evolutionary imperative is often to conserve resources for her own survival while providing sufficient nourishment to current and subsequent litters.
Accordingly, paternally expressed genes tend to be growth-promoting whereas maternally expressed genes tend to be growth-limiting.
In support of this hypothesis, genomic imprinting has been found in all placental mammals, where post-fertilisation offspring resource consumption at the expense of the mother is high.
In vitro culture and imprinting
In vitro culture and imprinting: DNA methylation
(KSOMaa, Global, Human Tubal Fluid, Preimplantation 1/Multiblast, and G1v5PLUS/G2v5PLUS) in relation to a best-case (in vivo-derived embryos) and a worst-case (Whitten culture) scenario. Imprinted DNA methylation and expression were examined at three well-studied loci, H19, Peg3, and Snrpn, in mouse embryos cultured from the 2-cell to the blastocyst stage. We show that embryo culture in all commercial media systems resulted in imprinted methylation loss compared to in vivo-derived embryos, although some media systems were able to maintain imprinted methylation levels more similar to those of in vivo-derived embryos in comparison to embryos cultured in Whitten medium. However, all media systems exhibited loss of imprinted H19 expression comparable to that using Whitten medium. Combined treatment of superovulation and embryo culture resulted in increased perturbation of genomic imprinting, above that from culture alone, indicating that multiple ART procedures further disrupt genomic imprinting. These results suggest that time in culture and number of ART procedures should be minimized to ensure fidelity of genomic imprinting during preimplantation development.
For the Beckwith- Wiedemann syndrome, what are the two important genes in this domain and tell me about each
Beckwith-Wiedemann
The important genes in this domain are insulin-like growth factor 2 (IGF2) and H19.
IGF2 is expressed from the paternal allele, and the gene product has an important role in development and growth,
whereas H19 is a maternally expressed, non-coding RNA, which may function as a tumour suppressor, but whose precise biological role remains unresolved.
In ∼5% of BWS patients, gain of DNA methylation occurs on the normally unmethylated maternal H19DMR (H19DMR-GOM)
Overexpression of IGF2 and reduced expression of H19 give rise to the phenotype.
What are some other environmental factors in the oocyte and sperm ?
Strong experimental data also link paternal preconception nutrition with pathophysiology in the offspring, but the mechanism(s) routing effects of paternal exposures remain elusive. Animal experimental models have highlighted small non-coding RNAs (sncRNAs) as potential regulators of these effects. This study characterised the baseline sncRNA landscape of human sperm and the effect of a 6-week dietary intervention on their expression profile. 5’tRFs (tRNA derived fragments), miRNA and piwi interacting RNAs piRNAs were the most abundant sncRNA subtypes identified; their expression was associated with age, BMI and sperm quality. Nutritional intervention with vitamin D and omega-3 fatty acids altered expression of 3 tRFs, 15 miRNAs and 112 piRNAs, targeting genes involved in fatty acid metabolism and transposable elements in the sperm genome.
report on the effects of restricting the supply of specific B vitamins (i.e., B (12) and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep.
DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups.
no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure-effects that were most obvious in males.
The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the foetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring and modify adult health-related phenotypes.
How does a zinc deficiency effect the oocyte?
Impairs oocyte maturation, fertilisation and blastocyst development (in vitro and in vivo)
Alters DNA methylation, histone modification and imprinted gene expression
Whats the rescue strategy to target those individuals specifically with epigenetic (methylation) deficiencies?
