Blastulation = formation of a hollow ball of cells (blastocyst in mammals).

Mammalian Blastocyst (approximately day 6–7 after fertilization in the cow):
• Outer trophoblast → contributes to chorion and the fetal placenta; other fetal membranes also include epiblast-derived tissues.
• Inner Cell Mass (ICM/Embryoblast) → forms the embryo proper.
• Blastocoel — fluid-filled cavity.
• Still surrounded by zona pellucida; 'hatches' from zona before implantation (cow ~day 9).

Avian / Reptilian: A flat blastoderm forms on the yolk surface over a subgerminal cavity; it differentiates into epiblast and hypoblast rather than forming a spherical mammalian blastocyst.

Frog (Amphibian): Hollow ball with floor (vegetal yolk cells) thicker than roof (animal cap) → asymmetric blastula.

BLASTULATION — FORMATION OF THE BLASTOCYST

Blastulation is the formation of a hollow blastula stage from the morula. In mammals the resulting structure is called the blastocyst.

I. MAMMALIAN BLASTOCYST FORMATION (CAVITATION):
1. Tight junctions form between outer cells of morula → seal the embryo.
2. Na⁺/K⁺ ATPase pumps in outer cells pump Na⁺ into intercellular spaces.
3. Water follows osmotically → forms a fluid-filled cavity = BLASTOCOEL.
4. Inner cells cluster at one pole = Inner Cell Mass (ICM) / Embryoblast.
5. The EMBRYONIC POLE = side with ICM. Opposite side = ABEMBRYONIC POLE.

BLASTOCYST COMPONENTS:
• Trophoblast (Trophectoderm): outer cell layer; forms placenta + foetal membranes (chorion). 2 sub-types after implantation:
– Cytotrophoblast — inner mononuclear layer.
– Syncytiotrophoblast — outer multinucleate fused cells; invades endometrium.
• Inner Cell Mass (ICM): pluripotent cells; forms embryo proper + amnion + yolk sac + allantois.
• Blastocoel: fluid cavity.
• Zona pellucida: still surrounds blastocyst — must be shed before implantation.

HATCHING:
The blastocyst secretes proteases (e.g. plasminogen activator) to digest the zona pellucida and 'hatches' from it. Necessary for implantation. Failure to hatch is a cause of early embryonic loss in IVF.
• Cow: hatches ~day 9.
• Bitch: ~day 12.
• Mare: ~day 7-8 (mare also keeps an extra capsule for ~3 weeks).

II. ICM DIFFERENTIATION:
The ICM splits into two layers before implantation:
• Epiblast (dorsal layer of ICM) → contributes to ALL three germ layers of embryo + amnion.
• Hypoblast (ventral layer, facing blastocoel) → contributes mainly to YOLK SAC lining; part of the early endoderm.
This 2-layered disc is the BILAMINAR EMBRYONIC DISC.

III. AVIAN/REPTILIAN BLASTULATION (different):
Because of the large yolk, a flat disc of cells (blastoderm) sits on top of yolk, splitting into:
• Epiblast (top).
• Hypoblast (bottom, contributes to yolk-sac endoderm).
The space between them = blastocoel.

IV. AMPHIBIAN BLASTULA:
Hollow ball; the floor (large yolk-laden vegetal cells) is thicker than the roof (small animal cap cells). Marginal zone in between is where gastrulation begins.

V. SIGNIFICANCE OF BLASTOCYST:
• Defines the first cell-fate decision (trophoblast vs ICM).
• Enables maternal recognition of pregnancy (trophoblast secretes species-specific signals such as interferon-tau in cattle; in the mare, conceptus mobility and suppression of endometrial prostaglandin release are essential, while the definitive signal remains incompletely identified (eCG is produced later by endometrial cups)).
• Sets stage for implantation.
• Can be biopsied for genetic testing (PGD/PGS).

VI. STEM CELLS:
• Embryonic Stem Cells (ESC) are derived from ICM of a mammalian blastocyst → pluripotent.
• Used in research and veterinary regenerative medicine.