Quartz 5

02 - Fertilization & First Week of Human Development

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TARGET DECK: MED::I::Morphology and Development::Embryology::02 - Fertilization & First Week of Human Development

Embryology — Lesson II: Fertilization and Week 1

Prof. Mattia Lauriola

Ovulation

The follicle continues to enlarge and soon forms a bulge on the surface of the ovary. A small oval, avascular spot, the stigma, soon appears on this bulge.

Before ovulation, the secondary oocyte and some cells of the cumulus oophorus detach from the interior of the distended follicle.

The expelled secondary oocyte is surrounded by:

  • the zona pellucida — an acellular glycoprotein coat
  • one or more layers of follicular cells, radially arranged to form the corona radiata and cumulus oophorus

Timeline of Oocyte Maturation (hours from LH/FSH surge onset)

Time (h)Event
0Ovulatory surge of LH and FSH
~15Germinal vesicle breaks down
~20First meiotic metaphase; chromosomes line up → secondary oocyte + 1st polar body
~35Second meiotic metaphase begins
~38Arrest at second meiotic metaphase; ovulation follows

Ovulation Trigger

Ovulation follows within 24–36 hours of a surge of LH, which appears to result from signaling molecules from the granulosa cells (feedback). This surge, elicited by a high estrogen level in the blood, causes the stigma to rupture, expelling the secondary oocyte along with follicular fluid. Plasmins and matrix metalloproteinases (MMPs) also appear to have some control over stigma rupture.

Menstrual Cycle

The cycle is the period during which the oocyte matures, is ovulated, and enters the uterine tube.

Estrogen and progesterone produced by the ovarian follicles and corpus luteum cause cyclic changes in the endometrium of the uterus. These monthly changes in the uterine lining constitute the menstrual cycle.

Hormonal Feedback Loop

Hypothalamus → Pituitary gland → releases FSH and LH → acts on ovarian follicles → Estrogen and progesterone → negative feedback to hypothalamus/pituitary.

Viability of Oocytes and Sperms

Viability Windows

  • Oocytes are usually fertilized within 12 hours of ovulation
  • Oocytes cannot be fertilized after 24 hours and degenerate shortly thereafter
  • Most sperms do not survive more than 24 hours in the female genital tract
  • Some sperms are captured in folds of the cervical mucosa and are gradually released

Info

Semen and oocytes can be frozen and stored for many years for use in assisted reproduction.

Fertilization

Development begins at fertilization when a sperm penetrates an oocyte to form a zygote.

The Zygote

  • A highly specialized, totipotent cell — able to differentiate into any cell type
  • Contains chromosomes and genes derived from both mother and father
  • Divides many times and is progressively transformed into a multicellular human being through cell division, migration, growth, and differentiation

Site of Fertilization

Important

The usual site of fertilization is the ampulla, a dilation of the uterine tube.
If the oocyte is not fertilized, it slowly passes along the tube into the uterine cavity, where it degenerates and is resorbed.

Fertilization is a complex sequence of coordinated molecular events beginning with contact between a sperm and an oocyte. It ends with the intermingling of maternal and paternal chromosomes at metaphase of the first mitotic division of the zygote.

Sperm Chemotaxis

In vitro evidence suggests that the ovulated follicle releases a currently unknown sperm chemotropic factor, and that only capacitated sperm are able to respond to it by directed swimming toward the mature oocyte.
Carbohydrate- and protein-binding molecules on the surface of the gametes are involved in sperm chemotaxis, gamete recognition, and fertilization.

Phases of Fertilization

Phase 1 — Passage Through the Corona Radiata

Dispersal of follicular cells of the corona radiata results mainly from the action of the enzyme hyaluronidase, released from the acrosome of the sperm. Tubal mucosal enzymes also assist. Movements of the sperm tail are also important during penetration.

Phase 2 — Penetration of the Zona Pellucida

Enzymes released from the acrosome lyse the zona pellucida, forming a path for the sperm. Key enzymes include:

  • Acrosin (proteolytic)
  • Esterases
  • Neuraminidase

ZP3 Receptor Binding

When a spermatozoon reaches the zona pellucida, it binds in a species-specific interaction with the glycoprotein sperm receptor ZP3 (one of three glycoproteins composing the zona pellucida). This binding is mediated by the sperm surface protein SED1.
Binding involves a sequence of sugar molecules called sialyl-Lewis at the ends of the oligosaccharides of the ZP proteins.
Binding to ZP3 induces the acrosome to release degradative enzymes, allowing zona penetration.

Additional Membrane Receptors Involved

  • CD9 (tetraspanin) on the oocyte membrane binds IZUMO (IgG superfamily) from the sperm
    • IZUMO was named after the Japanese shrine to marriage
  • Integrin on the egg binds FERTILIN-beta on the sperm
  • Several factors from the ADAM superfamily (extracellular matrix proteins) are also involved

Phase 3 — Fusion of Plasma Membranes & Prevention of Polyspermy

Fusion of the plasma membranes of the oocyte and sperm occurs. The head of the sperm then enters the oocyte cytoplasm, but the plasma membrane and mitochondria of the sperm remain behind.

Upon fusion, two immediate events occur:

  1. A calcium wave radiates over the surface of the egg from the point of sperm contact
  2. Release of the contents of cortical granules (located just beneath the oocyte membrane) into the perivitelline space (between oocyte and zona pellucida)

Prevention of Polyspermy

The cortical granule contents alter the sperm receptor molecules, making the zona impenetrable to additional spermatozoa — this is called the zona reaction or block to polyspermy.
The trigger for cortical granule exocytosis is the release of ions from cortical smooth endoplasmic reticulum in response to sperm binding.

Contents and Actions of Cortical Granules

ComponentAction
ProteasesClip perivitelline tether proteins
PeroxidasesHarden the vitelline envelope
GlycosaminoglycansAttract water into perivitelline space → expand it → form the hyaline layer

Phase 4 — Completion of the Second Meiotic Division

The oocyte completes the second meiotic division, forming:

  • A mature oocyte
  • The second polar body

The nucleus of the mature oocyte becomes the female pronucleus.

Phase 5 — Formation of the Male Pronucleus

Within the oocyte cytoplasm, the sperm nucleus enlarges to form the male pronucleus. The tail of the sperm degenerates. During growth, both male and female pronuclei replicate their DNA.

The nuclear membranes quickly disappear as maternal and paternal chromosomes are replicated in preparation for the first cleavage.

Phase 6 — Breakdown of Pronuclear Membranes

  • Condensation of chromosomes
  • Arrangement for mitotic cell division
  • First cleavage division of the zygote

The combination of 23 chromosomes in each pronucleus results in a zygote with 46 chromosomes.

Results of Fertilization

Summary of Fertilization Outcomes

  • Stimulates the secondary oocyte to complete the second meiotic division, producing the second polar body
  • Restores the normal diploid number of chromosomes (46) in the zygote
  • Results in variation of the human species through mingling of maternal and paternal chromosomes

Reference links:

Chimeric Contribution of Human EPSCs to Monkey Embryos

Research Context

Human extended pluripotent stem cells (EPSCs) do not consistently and robustly contribute to chimera formation when the host animal has a high evolutionary distance from humans. Xenogeneic barriers between hPSCs and evolutionarily distant host animal species account for limited chimerism.

Reference: Cell. 2021 Apr 15;184(8):2020-2032.e14. doi: 10.1016/j.cell.2021.03.020

Cleavage of the Zygote

Info

Cleavage consists of repeated mitotic divisions of the zygote, resulting in a rapid increase in the number of cells called blastomeres.

  • Division begins approximately 24–30 hours after fertilization
  • Blastomeres become smaller with each cleavage division
  • The embryo does not increase in size during cleavage
  • The embryo remains enclosed in the zona pellucida throughout

Cleavage Timeline

Time Post-FertilizationStage
~24–30 hFirst cleavage → 2 blastomeres
~40 hSecond division → 4 equal blastomeres
~3 days6–12 cells
~4 days16–32 cells → morula

Compaction

After the eight-cell stage, blastomeres change shape and tightly align against each other — a process called compaction.

Compaction Details

  • Mediated by cell surface adhesion glycoproteins
  • Blastomeres flatten and develop inside-outside polarity, maximizing cell-to-cell contact
  • Outer cell surfaces become convex; inner surfaces become concave
  • Involves changes in the blastomere cytoskeleton
  • Prerequisite for segregation of the internal cells that form the inner cell mass

Embryoblast Formation

The inner cells of the morula — the embryoblast (inner cell mass, ICM) — are surrounded by a layer of flattened blastomeres forming the trophoblast.

Hippo signaling is an essential factor in segregating the ICM from the trophoblast.

Hippo Signaling Pathway

Mechanism

The Hippo pathway is a signaling cascade that regulates the nuclear localization of the transcriptional coactivator YAP.

  • Hippo ON (inner cells): YAP is phosphorylated → remains in cytoplasm → trophoblast fate suppressed
  • Hippo OFF (outer cells): YAP localizes to the nucleus → acts as co-transcription factor → trophoblast fate
FeatureOuter cellsInner cells
Lats1/2 kinasesAt contact-free surfacesMostly cytoplasmic
YAP localizationNucleusCytoplasm (phosphorylated)
AmotAt contact-free surfacesPhosphorylated, enriched at all surfaces; high expression
E-cadherin role—Excludes YAP from nucleus

Important

Amot only colocalizes with E-cadherin within inner cells, where its function is required, because Amot is excluded from basolateral surfaces in outer cells.

Biological Relevance of Hippo Pathway

Precise control of organ size is crucial during development and regeneration. Dysregulation leads to massive tissue overgrowth. Example: when two-thirds of a mouse liver is surgically removed, the remaining one-third regenerates its original mass within 7–10 days and then ceases growth.

Early Pregnancy Factor

An immunosuppressant protein — the early pregnancy factor — is secreted by trophoblastic cells and appears in maternal serum within 24–48 hours after implantation. It forms the basis for a pregnancy test applicable during the first 10 days of development.

Formation of the Blastocyst

About 4 days after fertilization, shortly after the morula enters the uterus, uterine fluid passes through the zona pellucida to form a fluid-filled blastocystic cavity inside the morula.

As fluid increases:

StructureOriginFate
TrophoblastThin outer cellsEmbryonic part of the placenta
Embryoblast (ICM)Central blastomeresEmbryo proper

Blastocyst Development

  • This stage is called blastogenesis; the conceptus is now called a blastocyst
  • The embryoblast projects into the blastocystic cavity; the trophoblast forms the wall
  • After ~2 days floating in uterine fluid, the zona pellucida degenerates and disappears (zona hatching)
  • Shedding permits the blastocyst to increase rapidly in size
  • While free-floating, the blastocyst derives nourishment from uterine gland secretions
  • The side containing the ICM is called the embryonic pole

Reference: https://studentconsult.inkling.com/read/larsen-human-embryology-schoenwolf-5/videos/animation-1-3

IVF (In Vitro Fertilization)

Indication

IVF is used when scarring of the oviducts (e.g., due to pelvic inflammatory disease, PID — a complication of sexually transmitted diseases such as gonorrhea) prevents sperm from reaching the ampulla or the fertilized oocyte from passing to the uterus.

IVF Procedure Overview

  1. Hormonal stimulation of multiple mature follicles
  2. Collection of oocytes from follicles by aspiration (laparoscopy or transvaginal)
  3. Placement of oocytes in a Petri dish with capacitated sperm → in vitro fertilization
  4. Cleavage of zygotes in culture medium until 4–8 cell stage
  5. Transfer of one or two cleaving embryos into the uterine cavity via catheter through vagina and cervical canal

Pharmacological Stimulation

  • FSH (sometimes combined with clomiphene citrate) is used for ovarian stimulation
  • Clomiphene citrate blocks hypothalamic estrogen detection → hypothalamus perceives estrogen deficiency → signals pituitary to release high FSH → stimulates follicle growth and estrogen secretion
  • Once estrogen rises sufficiently, the pituitary rapidly releases LH → oocyte maturation
  • hCG may also be given when follicles reach optimal size (monitored by ultrasound and plasma estradiol)

Reference links:

TLDR

  • Ovulation is triggered by an LH surge (elicited by high estrogen) 24–36 h beforehand; plasmins and MMPs contribute to stigma rupture
  • The secondary oocyte is arrested at second meiotic metaphase ~3 h before ovulation; meiosis II only completes upon fertilization
  • Oocytes are viable for ~12 h (max 24 h); most sperms survive <24 h in the female tract
  • Site of fertilization: ampulla of the uterine tube
  • Fertilization phases:
    • Phase 1: hyaluronidase disperses corona radiata
    • Phase 2: acrosin + esterases + neuraminidase lyse zona pellucida; ZP3 binds SED1; CD9↔IZUMO and integrin↔FERTILIN-beta mediate membrane docking
    • Phase 3: membrane fusion → Ca²⁺ wave → cortical granule exocytosis → zona reaction → block to polyspermy; sperm mitochondria stay outside
    • Phase 4: oocyte completes meiosis II → female pronucleus + 2nd polar body
    • Phase 5: sperm nucleus enlarges → male pronucleus; both pronuclei replicate DNA
    • Phase 6: pronuclear membranes break down → first cleavage → zygote with 46 chromosomes
  • Cleavage = repeated mitotic divisions without cell growth; embryo stays enclosed in zona pellucida
  • Morula reached at ~4 days (16–32 cells)
  • Compaction at 8-cell stage: blastomeres flatten and polarize via adhesion glycoproteins
  • Embryoblast (ICM) vs trophoblast segregation is governed by the Hippo/YAP pathway
    • Outer cells: YAP nuclear → trophoblast fate
    • Inner cells: YAP cytoplasmic (phosphorylated) → ICM/embryoblast fate
  • Blastocyst forms ~4–5 days post-fertilization; zona pellucida hatches ~2 days after uterine entry
  • Early pregnancy factor appears in maternal serum within 24–48 h of implantation (trophoblast-secreted)
  • IVF indications include blocked oviducts; uses FSH ± clomiphene citrate ± hCG for stimulation; 1–2 embryos transferred at cleavage stage

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