Internal Fertilization in Mammals

Overview

Site of Fertilization

Sperm Transport and Viability

• Of 200–300 million spermatozoa deposited in the vagina at ejaculation, only 300–500 reach the ampulla.
• Only 1% of sperm deposited in the vagina enter the cervix; cervical crypts provide short-term sperm storage and gradual release.
• Transit from cervix to uterine tube can take as little as 30 minutes or up to 6 days.
• Sperm remain viable in the female genital tract for up to 48 hours (some estimates up to several days).
• The oocyte must be fertilized within 12–24 hours of ovulation before it degenerates.

Pre-Fertilization Requirements

1. Capacitation

• A glycoprotein coat and seminal plasma proteins are removed from the acrosomal region of the plasma membrane.
• Capacitation primarily occurs via epithelial interactions with the mucosal lining of the uterine tube.
• Only capacitated sperm can pass through the corona radiata and undergo the acrosome reaction.

2. Acrosome Reaction

Phases of Fertilization

Events of fertilization: A–capacitation, B–acrosome reaction, C–membrane fusion, D–cortical reaction (Ca²⁺ wave, block to polyspermy), E–pronuclei formation and zygote. — The Developing Human, Fig. 2.13

Phase 1 — Penetration of the Corona Radiata

Phase 2 — Penetration of the Zona Pellucida

Phase 3 — Fusion of Oocyte and Sperm Cell Membranes

• Initial adhesion is mediated by integrins on the oocyte and disintegrins on the sperm.
• Sperm surface proteins — IZUMO, ADAMs 1/2/3, CRISP1 — bind to oocyte receptors (Juno, integrins, CD9, CD81).
• The plasma membranes fuse (at the posterior region of the sperm head, since the acrosomal membrane was lost).
• The sperm head and tail enter the oocyte cytoplasm; the plasma membrane of the sperm remains on the oocyte surface.

Oocyte Responses After Sperm Entry

Fertilization stages A–F: from secondary oocyte through two-cell zygote. — Langman's Medical Embryology, Fig. 3.6

1. Cortical and zona reactions — Lysosomal enzymes from cortical granules are released into the perivitelline space → the oocyte membrane becomes impenetrable to other sperm, and ZP2/ZP3 receptors are inactivated. This prevents polyspermy.
2. Resumption of the second meiotic division — The oocyte (arrested in metaphase II) completes meiosis II → produces the mature oocyte plus the second polar body. The oocyte nucleus becomes the female pronucleus (22 + X chromosomes).
3. Metabolic activation of the egg — The activating factor (likely phospholipase C-zeta from the sperm) triggers a Ca²⁺ wave that reactivates cell cycling and initiates the molecular events of early embryogenesis.

Pronucleus Formation and Syngamy

• The sperm nucleus decondenses and enlarges to form the male pronucleus; the tail detaches and degenerates.
• Male and female pronuclei are morphologically indistinguishable.
• Both pronuclei replicate their DNA (each haploid → 1n, 2c).
• The pronuclei come into close contact, lose their nuclear envelopes, and chromosomes organize on the cleavage spindle.
• This fusion event — syngamy — produces the zygote, restoring the diploid number (2n = 46 in humans).
• The oocyte containing two pronuclei just before this fusion is technically called an ootid.

Biological Results of Fertilization

Key Molecular Players

• The Developing Human: Clinically Oriented Embryology, 10th ed., pp. 116–128
• Langman's Medical Embryology, 14th ed., pp. 58–61

Internal Fertilization in Mammals

1. Sperm Transport to the Site of Fertilization

• Sperms pass through the cervical canal by flagellar movement; cervical crypts store some sperm and release them gradually, prolonging the fertilization window.
• Passage through the uterus into the uterine tube occurs mainly by muscular contractions of the uterus and tube walls — not primarily by sperm propulsion alone.
• Prostaglandins in semen stimulate uterine motility at the time of intercourse.
• Sperm move 2–3 mm per minute; motile sperm have been recovered from the ampulla as early as 5 minutes after deposition at the cervix, though transport can take up to 45 minutes.
• Sperm survive up to 48 hours in the female tract; the oocyte must be fertilized within 12–24 hours of ovulation.
• Of 200–300 million spermatozoa deposited, only 300–500 reach the site of fertilization. Only one fertilizes the egg — the rest likely assist in penetrating barriers surrounding the oocyte.

2. Pre-Fertilization Conditioning

Capacitation

• A glycoprotein coat and seminal plasma proteins are removed from the sperm acrosomal membrane.
• Cholesterol (the major inhibitor of capacitation) is stripped from the plasma membrane; phospholipid redistribution follows.
• Adenylyl cyclase activity increases → rising intracellular cAMP levels.
• Tyrosine phosphorylation (via Src kinase family) increases — used clinically as a biochemical marker of capacitation.
• Ca²⁺ channel activation → hyperpolarization → acquisition of hyperactivated motility (vigorous, whiplash-like flagellar beating driven by CatSper Ca²⁺ channels in the sperm tail).
• Capacitated sperm show no visible morphologic change but are far more active.

Acrosome Reaction

• Fusion of the sperm plasma membrane with the outer acrosomal membrane → formation of perforations (apertures).
• Release of acrosomal enzymes: hyaluronidase, acrosin (a serine protease), esterase, and neuraminidase.
• This is regulated by tyrosine kinase (Src kinase), calcium ions, prostaglandins, and progesterone.

3. The Three Phases of Fertilization

Phase 1 — Penetration of the Corona Radiata

• Hyaluronidase released from the acrosome.
• Tubal mucosal enzymes.
• Mechanical action of the sperm tail.

Phase 2 — Penetration of the Zona Pellucida

• Zona reaction: cortical granule enzymes (proteases) cross-link zona proteins and degrade ZP2/ZP3 receptors → zona becomes impermeable to further sperm penetration → permanent block to polyspermy.

Phase 3 — Fusion of Oocyte and Sperm Cell Membranes

• Initial adhesion: integrins on the oocyte bind to disintegrins on the sperm.
• Key fusion proteins: sperm IZUMO1 binds oocyte Juno (essential for membrane fusion); also fertilin (PH-30), ADAMs 1/2/3, CRISP1 interact with oocyte CD9, CD81, and integrins.
• Fusion occurs at the posterior sperm head (the acrosomal membrane was shed during the acrosome reaction).
• The sperm head and tail enter the oocyte cytoplasm; the plasma membrane remains as a surface appendage; sperm mitochondria are excluded (all mitochondria in the embryo are maternal).
• The paternal centrosome is incorporated — it is essential for building the first and subsequent mitotic spindles.

4. Oocyte Responses After Sperm Entry

i. Block to Polyspermy (two sequential mechanisms)

ii. Resumption of the Second Meiotic Division

• Produces the mature oocyte + second polar body (expelled into the perivitelline space).
• The oocyte nucleus becomes the female pronucleus (22 + X chromosomes).
• Signaled by phospholipase C-zeta 1 (PLCζ1) from the sperm, which triggers sustained Ca²⁺ oscillations in the oocyte cytoplasm, reactivating cell cycling.

iii. Metabolic Activation

5. Pronucleus Formation and Syngamy

Stages of fertilization from secondary oocyte (A) to two-cell zygote (F). — Langman's Medical Embryology, Fig. 3.6

• The sperm nucleus decondenses and enlarges → male pronucleus (23 paternal chromosomes); the sperm tail degenerates.
• Male and female pronuclei are morphologically indistinguishable.
• Both pronuclei replicate their DNA (each 1n → 1n, 2c).
• The oocyte containing two pronuclei at this stage is technically called an ootid.
• Pronuclei migrate toward the center, nuclear envelopes dissolve (without fusing), and chromosomes align on the first mitotic (cleavage) spindle.
• This event — syngamy — produces the zygote: 2n = 46 chromosomes in humans.

A: Pronuclear stage of fertilized human oocyte. B: Two-cell stage. — Langman's Medical Embryology, Fig. 3.7

6. Outcomes of Fertilization

7. Key Molecular Players Summary

• The Developing Human: Clinically Oriented Embryology, 10th ed., pp. 111–128
• Langman's Medical Embryology, 14th ed., pp. 58–61
• Histology: A Text and Atlas with Correlated Cell and Molecular Biology, pp. 2221–2226