Mammalian Reproductive Biology - Midterm Exam

Epigenetic change

Chemical modification on DNA or chromatin structure that alters the function of DNA without changing the actual DNA sequence. Germ cells (eggs and sperm) can carry these modifications and pass them onto offspring, which can affect generations of offspring.

What are the three major epigenetic markers?

1. DNA Methylation

2. Histone modification

3. Micro RNAs

What are introns?

Non-coding regions of the RNA strand; are not translated into proteins and need to be cut out. 

Why are histones attracted to the DNA strand?

The phosphodiester bonds of the DNA are negatively-charged, which attracts the positive charges on histone proteins. 

Chemical mechanism of DNA methylation 

• catalytic reaction to create 5’-Methylcytosine 

• DNMT adds a methyl group to cytosine using the methylating agent SAM → methyl group binds to the C5 region of the cytosine group

DNMT

DNA methyltransferase; responsible for adding a methyl group to DNA 

CpG strand

A repetitive sequence of C and G bases; the region where DNA methylation takes place 

SAM

S-adenosylmethionine → supplies a methyl group to cytosine during DNA methylation, and is turned into SAH (S-adenosylhomocysteine) after the reaction

HAT

Histone acetyltransferases → enzymes that acetylate conserved lysine amino acids

• allow the DNA to be in a relaxed state → transcription factors able to bind 

Methylation of globin genes

• at 6 weeks of embryonic growth, the Epsilon(E)-globin promoter is unmethylated and active

• at 12 weeks of growth, the E-globin promoter is methylated (inactive), and the gamma(y)-globin promoter is unmethylated (active) 

• this is because at 6 weeks, the bone marrow is not fully formed yet, so the liver makes hemoglobin; after 12 weeks, the bone marrow is able to create hemoglobin, so the E-globin promoter for the liver is switched off 

HDAC

Histone Deacetylase → enzyme that removes acetyl groups from proteins, which can lead to gene repression

• causes the DNA to tighten, so transcription factors are unable to bind

What are nucleosomes?

Units comprised of DNA wrapped around histone proteins

Linker DNA

The region of DNA between two connected histones

• Histone 1 (H1) is the Linker → attracts other H1s to attract histones to each other, tightening the DNA strand

Methylation on Histone H3

• depending on the position of the lysine on the tail, this activates or deactivates the histone

• specific protein binding to H3 histone tails determines whether it will be methylated or unmethylated

MeCP2

• Methyl-CpG Binding Protein 2

• Binds to methylated cytosine residues in DNA, helping to silence gene expression

• MECP2 gene is located on the X chromosome

HMT

histone methyltransferase → binds to histone tail and adds a methyl group; works jointly with MeCP2

Activation vs. Repression of Histones

• Activation = H3K4me3, H3K36me3, H3 acetylation

• Repression = H3K9me3, H3K27me3

What are the two checkpoints for developmental epigenetic programming?

1. The first checkpoint occurs around 1-2 weeks post conception (from the formation of the blastocyst to implantation)

2. The second checkpoint occurs during sex determination

These checkpoints represent windows when DNA methylation patterns are erased and re-established, making them particularly vulnerable to environmental influences

RNAPII

RNA Polymerase II; a multiprotein complex that transcribes DNA into precursors of mRNA and most small nuclear RNA (snRNA) and miRNA

• RNAPII expressed = open chromatin → transcription ON

PRC2

• Polycomb Repressive Complex 2

• catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3), leading to the compaction of chromatin and the repression of gene transcription

• suppression of this protein complex is linked to developmental disorders and various cancers

Where do cells with the highest morphogen concentration go?

Towards the center

Are epigenetic changes inherited through germ cells or somatic cells?

Germ cells, since they are able to be passed down to offspring. They cannot be passed on through somatic cells (all other body cells), as those are limited to the individual organism. 

What are the three types of cell signaling?

1. Autocrine = a cell sends signals to target itself

2. Juxtacrine = short-range signaling (direct contact between cells)

3. Paracrine = long-range signaling (signals travel through extracellular space)

What do the position of cell aggregates depend on?

The surface tension (force/length) of each cell type

What is surface tension of cells determined by?

The presence of cell-adhesion molecules, such as proteins like cadherins

What happens when cells have the same surface tension?

When surface tension is the same, cells form boundaries by replacing cadherin with myosin. 

Promoter Region 

When methylated, transcription factors cannot bind → gene is silenced 

Enhancer region

Located upstream of promoter; increases transcription likelihood when active

• when methylated → decreased gene transcription

• enhancer affinity influences promoter activity

What is the structure of chromatin?

147 base pairs of DNA wrapped around histones to form nucleosomes 

Composition of a histone octamer

The protein core, composed of two each of H2A, H2B, H3, and H3. DNA wraps around this core, with the linker histone H1 binding to the linker DNA outside the core to further stabilize the structure.

Euchromatin

Relaxed/open state of chromatin → genes are accessible for transcription

Heterochromatin

Condensed/closed state of chromatin → genes are inaccessible and silenced

Function of microRNAs

Small RNAs that silence protein synthesis by binding to mRNA and segmenting it to degrade the strand of mRNA, which prevents translation

Regulation of miRNA

Produced in response to excessive mRNA levels → miRNA will break down the excess mRNA to prevent the overproduction of proteins

What is the clinical significance of miRNAs?

They can serve as biomarkers for disease states 

Epigenetic checkpoint 1

Occurs 1-2 weeks post-conception

• Occurs after blastocyst formation

• Epigenetic changes will affect the somatic (body) cells of the developing embryo

Epigenetic checkpoint 2

Occurs during sex differentiation

• Epigenetic changes will affect the germ line (reproductive cells)

• Therefore, these alterations are transgenerational, meaning they can be passed on to the offspring

What is the high susceptibility period?

Between 3-8 weeks gestation 

• different organs have critical developmental windows

How is immune cell differentiation determined?

It is guided by morphogen gradients

What can chemical exposures disrupt?

1. Morphogen gradients

2. Cell differentiation phases

3. Organ development

Long term effects of epigenetic changes

• DNA methylation changes during development can persist throughout life

• dysregulated genetic marks can be activated OR regulated marks can be repressed

• These changes can affect future generations through germ line modifications

Surface tension and cell sorting

• Cells with lower surface tension surround cells with higher surface tension

• this differential adhesion drives cell sorting and tissue organization

What are cadherins?

Cell adhesion molecules 

• form cadherin-cadherin bonds between adjacent cells 

• critical for maintaining tissue structure 

What do integrins do?

They link cells to the extracellular matrix (ECM) and are essential for cell survival.

What would happen if integrins fail to bind to the ECM?

• Cell-cell communication will be disrupted

• Cells will not be able to survive, and will undergo apoptosis

• Loss of actin cytoskeleton support

• Cells may undergo epithelial-mesenchymal transition (EMT)

Function of actin filaments

Required for maintaining structural integrity of the cell → function as “anchors”

Inside the cell, ____ interact with cadherins and bind to the actin cytoskeleton (microfilaments). This bonding keeps the cells together.

Catenins

Epithelial-Mesenchymal Transition (EMT)

The process in which epithelial cells lose their polarity and adhesion, gaining migratory and invasive properties.

What kind of signaling is involved with EMT?

Paracrine signaling 

Why is the epithelial-mesenchymal transition related to cancer?

Cancer cells use EMT to migrate and find oxygen-rich areas for survival.

• offering more oxygen can be used as a way to keep the cancer cells in place long enough for them to be treated

Functions of morphogen gradients

• Create concentration gradients across developing tissues

• Tell cells their position in the developing embryo

• Position determines cell fate and differentiation

Structure of Morphogen Gradients 

• High concentration = cells migrate toward the center/source 

• Low concentration = cells are at the periphery/edges 

How can the disruption of morphogen gradients lead to birth defects?

• Dysregulated gradients prevent proper differentiation

• Changing gradients can cause abnormal cell positioning

• Loss of proper cell-cell communication

What is a potential consequence of inner cell mass changes of the morphogen gradient?

Cells may repel each other, causing two separate cells masses on either side of the blastocyst → results in twin formation

What is a morphogen?

A diffusible biochemical molecule (paracrine) that can determine the fate of a cell by its concentration 

Signal transduction cascades

• Every cell has at least one type of receptor for morphogens

• Cascades have master regulators that control downstream responses

  • If a master molecule is inhibited, the ENTIRE cascade will be disrupted

RTK Signaling Pathway 

RTK = Receptor Tyrosine Kinase 

• Morphogens regulate differentiation through activation of RTK

• Sequential phosphorylation events amplify the signal

What are four major signaling pathways?

1. JAK-STAT Pathway

2. WNT Pathway

3. SMAD Pathway

4. Notch Pathway

JAK-STAT Pathway

• Function = Immune response regulation

• Ligand binding activates JAK kinases → STAT transcription factors

What is the function of the WNT pathway?

Female sex development and organ development

• WNT4 knockout → leads to impaired kidney development

What is the mechanism for the WNT pathway?

1. WNT ligand inhibits GSK3

2. Beta-catenin accumulates and enters the cell nucleus

3. Activates target gene expression

SMAD Pathway

• Function: Cell growth, division, and development

• Activated by TGF-beta superfamily ligands

Notch pathway 

• Function: Cell differentiation and fate determination 

• Requires direct cell-cell contact 

What would happen if a ligand or receptor is disrupted in the WNT signaling pathway?

• The embryo will fail to develop properly

• Gene expression will not occur

• Subsequent developmental responses will be blocked

Where does fertilization occur?

Ampulla of fallopian tube

What process must occur before fertilization?

Capacitation of sperm → removal of glycoprotein and seminal plasma coat

• sperm undergoes biochemical changes in the female reproductive tract 

Acrosome reaction 

First step of fertilization:

• Once bound to the zona pellucida, enzymes are released to help the sperm penetrate the oocyte.

• IZUMO protein is expressed on the sperm membrane

What are the basic steps of fertilization?

1. Acrosome Reaction

2. Penetration of Corona Radiata

3. Binding to Zona Pellucida

4. Prevention of Polyspermy

5. Izumo-Juno Interaction

6. Completion of Meiosis

Penetration of Corona Radiata 

Second step of fertilization: 

• Enzymes on the sperm head digest through the outer cell layer 

  • Use hyaluronidase acid enzymes

Binding to the Zona Pellucida

Third step of fertilization:

• Sperm binds to the glycoprotein layer surrounding an egg

• ZP3 protein 

What happens if the zona pellucida does not have enough oxygen (hypoxia)?

• It will become too hard, preventing sperm from binding or breaking through → may result in delayed implantation

• Can be reversed if oxygen is reintroduced

Prevention of polyspermy

Fourth step of fertilization:

• Critical to prevent multiple sperm from fertilizing one egg

• mechanisms: fast block (electrical) and slow block (cortical granule release)

What happens if polyspermy DOES occur?

The egg will become “invisible” due to too many chromosomes 

IZUMO-JUNO Interaction

Fifth step of fertilization: 

• IZUMO ligand on sperm binds to the JUNO receptor on egg

• This binding is required for membrane fusion → the Izumo protein mediates the adhesion and subsequent fusion of sperm with the egg cell membrane

Completion of Meiosis

Sixth step of fertilization:

• Egg completes meiosis II after fertilization

• Sperm prepares by removing flagella and comes closer to female pronucleus

• Then, both undergo mitotic division

Why does the corpus luteum stop producing progesterone after a while?

The embryo is able to start producing its own progesterone.

Hyaluronidase acid enzyme

Function: Digest the glycoprotein layer; helps with penetration into the corona radiata

Embryonic development timeline

• 0-3 weeks = early development (cleavage, gastrulation)

• 4-8 weeks = period of embryonic organogenesis

• 9-38 weeks = fetal period

What event marks “Day 1” of development?

Fertilization → wherein the sperm meets the egg in the fallopian tube

Events during Day 2 of development:

Cleavage continues → Two-cell stage

• Rapid mitotic divisions

• maternal mRNA degrades as the zygotic genome activates (able to produce its own mRNA)

Events during Day 3 of development 

Morula stage (16-32 cells; form a solid ball)

Events during Days 4-5 of development:

Early blastocyst forms on Day 4, while late blastocyst forms on day 5.

• fluid-filled cavity (blastocoel) develops

• two distinct cell populations form

Events during Day 7-7.5 of development:

• Two layers of the trophoblast form: Cytotrophoblast and Syncytiotrophoblast 

• Two layers of inner cell mass form: Hypoblast and Epiblast 

What two layers does the trophoblast split into?

1. Cytotrophoblast → Inner layer

2. Syncytiotrophoblast → Outer layer (invades the uterine lining)

What two layers does the embryoblast split into?

1. Epiblast (primitive endoderm) → eventually forms the three germ layers (endoderm, ectoderm, mesoderm)

2. Hypoblast → eventually forms the yolk sac and extraembryonic tissues 

What two layers does the extra-embryonic mesoderm split into?

1. Somatic layer (Parietal layer) → Forms the body wall structures 

2. Splanchnic layer (Visceral layer) → Forms the gut-associated structures 

What are the two cavities that form during embryonic development?

1. Yolk sac 

2. Amniotic cavities 

Events during Day 13 of development:

• Lacunar channels form in the syncytiotrophoblast → allow maternal blood to flow around the embryo

• Cytotrophoblast maintains structural integrity

What does the mesoderm differentiate into?

• Bones

• Connective tissue

• Urogenital system

• Cardiovascular system

Maternal-fetal gas exchange 

• Occurs across the placental interface 

  • Oxygen and nutrients from the mother are passed to the fetus

  • Carbon dioxide and waste from the fetus are passed to the mother

Primordial Germ Cells (PGCs)

The precursors to all reproductive cells, first formed in the yolk sac during early embryonic development

What do the PGCs differentiate into?

• Males → spermatogonia (sperm stem cells)

• Females → oogonia (egg precursors)

What is the basic function of somatic cells in gonadal development?

They provide structural and nutritional support to germ cells 

What does the ectoderm differentiate into?

• Skin

• Central and peripheral nervous systems

• Eyes

• Internal ear

• Neural crest cells → bones and connective tissue of the face + part of the skull

What does the endoderm differentiate into?

The gut and gut derivatives (liver, pancreas, etc.)

What are the three domains of the ectoderm?

1. Surface ectoderm → primarily epidermis

2. Neural crest → peripheral neurons, pigment, facial cartilage

3. Neural tube → brain and spinal cord

What are the gonadal somatic cells in males?

1. Sertoli cells → support sperm development; secrete Anti-Mullerian Hormone (AMH)

2. Leydig cells → produce testosterone

What are the gonadal somatic cells in females?

1. Granulosa cells → support egg development

2. Theca cells → produce androgen precursors for estrogen synthesis

The Indifferent Stage 

Early embryos have TWO duct systems that can develop into either male or female reproductive tracts 

• Wolffian ducts (mesonephric ducts) → become male internal structures if preserved 

• Mullerian ducts (paramesonephric ducts) → become female internal structures if preserved 

Where did the Wolffian and Mullerian tubes develop from?

Both develop from the intermediate mesoderm along the genital ridge