Gametes and fertilization

Development begins before fertilization. The egg and sperm are among the most specialized cells an animal makes, and the egg in particular is stocked with the mRNAs, proteins, nutrients and organelles that will run the first stages of development, often before the embryo's own genes are switched on. Fertilization then accomplishes two things at once: it combines the genetic material of two parents, and it activates the egg, triggering the metabolic and cell-cycle events that start embryogenesis. This lesson follows gametes from their formation to the fusion of the two pronuclei, using the sea urchin and the mouse as the main examples, with human applications where they illuminate the biology.

Making gametes

Gametes arise from primordial germ cells, a lineage set aside early in development and discussed in lesson 10. After migrating to the developing gonad, germ cells proliferate and then enter meiosis, the two divisions that halve the chromosome number and shuffle parental alleles, as described in BIOL-107 lesson 12 and BIOL-201 lesson 6. Spermatogenesis and oogenesis differ strikingly in timing and in what they produce.

In mammalian spermatogenesis, spermatogonial stem cells in the seminiferous tubules divide throughout adult life. Some daughters enter meiosis as primary spermatocytes, and each gives rise to four haploid spermatids. The spermatids then undergo spermiogenesis, a dramatic remodelling in which the nucleus condenses as histones are replaced by protamines, the Golgi forms the acrosome, a centriole organizes the flagellum, mitochondria wrap around the midpiece, and most of the cytoplasm is discarded. The cells remain joined by cytoplasmic bridges until release, which allows haploid cells to share gene products. In humans the process takes about 74 days from stem cell to sperm, and a man produces on the order of a hundred million sperm per day.

Oogenesis is slower and more conservative. In mammals, oogonia proliferate only before birth, then enter meiosis and arrest in prophase of meiosis I as primary oocytes within follicles. A human female is born with roughly one to two million oocytes, of which only a few hundred will ever be ovulated; most degenerate. At puberty, hormonal cycles recruit follicles each month; the selected oocyte completes meiosis I, extruding a small first polar body, and arrests again at metaphase of meiosis II. It completes meiosis only if fertilized. The asymmetric divisions conserve cytoplasm: one large egg and up to three tiny polar bodies are produced, rather than four equal cells. This long arrest, decades in humans, is thought to contribute to the rise in chromosomal errors such as trisomy 21 with maternal age.

What the egg contains

Eggs vary in size from about 0.1 mm in mammals to 1.2 mm in Xenopus and several centimetres in birds, where most of the volume is yolk. Beyond yolk, the egg contains the maternal contribution to development. Maternal mRNAs, transcribed during oogenesis and stored in a translationally repressed state, encode proteins for the early cell cycles, signalling components and transcription factors. Some are localized in specific regions of the egg, where they act as cytoplasmic determinants: bicoid mRNA at the anterior of the Drosophila egg, VegT mRNA in the vegetal cytoplasm of Xenopus, and germ plasm in many species. The egg also contains ribosomes, mitochondria (all inherited from the mother in most animals), cortical granules beneath the plasma membrane, and protective coats: the vitelline envelope of sea urchins and the zona pellucida of mammals. Genes expressed by the mother that affect the embryo through these products are called maternal effect genes, because the phenotype of the embryo depends on the mother's genotype rather than its own.

Getting gametes together

Species with external fertilization, such as sea urchins, many fish and frogs, release gametes into water, where sperm must find eggs of their own species among those of others. Many marine eggs release chemoattractant peptides. The sea urchin Arbacia punctulata releases resact, which binds a receptor guanylyl cyclase on the sperm tail and steers swimming up the concentration gradient. Chemoattraction is species-specific, the first of several recognition steps.

In mammals, sperm deposited in the female tract are not immediately able to fertilize. They must undergo capacitation, a set of changes that take hours in the female tract and include removal of cholesterol and glycoproteins from the sperm membrane, increased membrane fluidity, phosphorylation of proteins and a change to a vigorous, whip-like hyperactivated motility. Capacitation is why in vitro fertilization requires incubating sperm in specific media. Only a few hundred of the millions of sperm in an ejaculate reach the oviduct, where fertilization occurs. Guidance there probably combines rheotaxis (swimming against fluid flow), thermotaxis toward the slightly warmer site of fertilization, and possibly chemotaxis, mediated in part by the sperm-specific CatSper calcium channel.

Recognition and the acrosome reaction

When a sea urchin sperm meets the jelly coat of an egg, sugar polymers carrying sulphate groups in the jelly trigger the acrosome reaction: the acrosomal vesicle fuses with the sperm plasma membrane and releases enzymes that digest a path through the jelly, and actin polymerization extends an acrosomal process. The process carries the protein bindin, which binds species-specifically to a receptor on the vitelline envelope. Bindin from one sea urchin species binds poorly to the eggs of another, and bindin sequences evolve rapidly under selection, contributing to reproductive isolation between species.

In mammals the sperm must penetrate the cumulus cells surrounding the egg and then the zona pellucida. The mouse zona is made of three or four glycoproteins, ZP1 to ZP3 in mice and ZP1 to ZP4 in humans. Current evidence favours a model in which the N-terminal region of ZP2 is the main sperm-binding site, and the acrosome reaction may often begin before the sperm reaches the zona, in the cumulus. After passing through the zona, the sperm fuses with the egg membrane. In 2005 Masaru Okabe's group identified the sperm protein Izumo1 as essential for fusion, and in 2014 Gavin Wright's group identified its egg receptor, Juno; knockout mice lacking either protein are infertile. More proteins required for gamete fusion continue to be discovered.

Fusion and blocks to polyspermy

In most animals, fertilization by more than one sperm, polyspermy, is lethal, because the extra centrioles and chromosomes disrupt mitosis. Eggs therefore have mechanisms that prevent more than one sperm from entering. Sea urchins have two. The fast block acts within one to three seconds: sperm fusion opens sodium channels in the egg membrane, and the membrane potential shifts from about −70 mV to about +20 mV. Sperm cannot fuse with a depolarized egg. Laurinda Jaffe showed in 1976 that holding sea urchin eggs at a negative potential by passing current allowed polyspermy, while depolarizing them prevented fertilization. The fast block is transient and is not used by mammals.

The slow block lasts. About 20 to 60 seconds after fusion, cortical granules beneath the egg membrane fuse with it and release their contents. Enzymes cleave the links between the vitelline envelope and the membrane, mucopolysaccharides draw water into the space and lift the envelope away, and a peroxidase hardens it into a fertilization envelope that sperm cannot penetrate. In mammals, the cortical reaction releases the protease ovastacin, which cleaves ZP2 so that the zona no longer binds sperm; Juno is also rapidly shed from the egg membrane after fertilization, which may help block membrane fusion.

Egg activation and the calcium wave

The cortical reaction is triggered by a wave of calcium that sweeps across the egg from the point of sperm entry. Free calcium rises from about 0.1 µM to around 1 µM, released from the endoplasmic reticulum through inositol trisphosphate (IP3) receptors. The wave crosses a sea urchin egg in about 30 seconds. Calcium is both necessary and sufficient for activation: injecting a calcium chelator blocks activation by sperm, while calcium ionophores activate unfertilized eggs, a phenomenon called parthenogenetic activation. In mammals, the sperm delivers a phospholipase, PLCζ, into the egg cytoplasm; it generates IP3 and drives a series of calcium oscillations that continue for hours. Human sperm lacking functional PLCζ cause failure of fertilization even with intracytoplasmic sperm injection, and artificial activation with calcium ionophore is used in some fertility clinics, a practice still being evaluated.

Calcium triggers the events of egg activation. Early responses include the cortical reaction and, in mammals, the completion of meiosis II and extrusion of the second polar body, as calcium leads to destruction of cyclin B and inactivation of maturation-promoting factor. Later responses, over minutes to hours, include a burst of protein synthesis from stored maternal mRNAs, an increase in intracellular pH in sea urchins, resumption of DNA synthesis and entry into the first mitotic cell cycle.

import math, random
random.seed(7)
# A calcium wave crossing an egg: a wavefront moving at constant speed v from the sperm entry point.
# Each patch of cortex releases its cortical granules when the wave arrives.
diameter_um = 100.0      # sea urchin egg diameter (approximate)
v = 5.0                  # wave speed, micrometres per second (illustrative, within the reported range)
# arrival time at points around the circumference, measured from the entry point (angle 0)
for deg in (0, 45, 90, 135, 180):
    arc = math.pi * diameter_um * deg / 360          # distance along the surface
    print(f"angle {deg:3d} deg: wave arrives after {arc / v:5.1f} s")
# fraction of cortex activated over time (uniform points on the circle)
pts = [random.uniform(0, 180) for _ in range(20000)]
for t in (5, 10, 20, 30):
    reached = sum(1 for d in pts if math.pi * diameter_um * d / 360 / v <= t) / len(pts)
    print(f"t = {t:2d} s: {100 * reached:5.1f} % of the cortex has released granules")

angle   0 deg: wave arrives after   0.0 s
angle  45 deg: wave arrives after   7.9 s
angle  90 deg: wave arrives after  15.7 s
angle 135 deg: wave arrives after  23.6 s
angle 180 deg: wave arrives after  31.4 s
t =  5 s:  16.0 % of the cortex has released granules
t = 10 s:  32.2 % of the cortex has released granules
t = 20 s:  63.9 % of the cortex has released granules
t = 30 s:  95.4 % of the cortex has released granules

The simulation reproduces the order of magnitude of the real event: a wave travelling at a few micrometres per second crosses a 100 µm egg in about 30 seconds, and the cortical reaction follows it around the egg. In real eggs the wave is self-propagating, because calcium released at one site promotes release at neighbouring sites through calcium-sensitive IP3 receptors, so its speed stays roughly constant rather than slowing with distance as simple diffusion would.

The pronuclei unite

After fusion, the sperm nucleus decondenses as protamines are replaced by maternal histones, and a male pronucleus forms. In mammals the egg also finishes meiosis to form the female pronucleus. The sperm centriole, in most animals, organizes a microtubule aster that brings the pronuclei together. In sea urchins the pronuclei fuse to form a diploid zygote nucleus. In mammals they do not fuse directly; instead, both replicate their DNA, their envelopes break down, and the chromosomes come together on the first mitotic spindle, so the two parental genomes first share a nucleus at the two-cell stage. Recent imaging has shown that mammalian zygotes may assemble two separate spindles, one around each parental genome, which then align, offering one explanation for the frequent errors in early human embryos.

The parental genomes are not equivalent. Genomic imprinting marks certain genes, by DNA methylation laid down during gametogenesis, so that only the maternal or only the paternal copy is expressed. Mouse embryos made with two maternal or two paternal genomes fail to develop, which is why mammals cannot normally reproduce parthenogenetically. Imprinted genes such as Igf2 (expressed from the paternal copy) influence growth and placental development, and errors in imprinting cause disorders such as Prader–Willi and Angelman syndromes. After fertilization, most of the genome is demethylated and then remethylated as the embryo develops, but imprinted regions are protected; this epigenetic reprogramming was introduced in BIOL-201 lesson 12.

Fertilization technology and its limits

Knowledge of fertilization underlies assisted reproduction. In vitro fertilization (IVF), first successful in humans with the birth of Louise Brown in 1978, relies on capacitating sperm in culture and on the fact that the egg completes meiosis on activation. Intracytoplasmic sperm injection (ICSI), introduced in 1992, bypasses the zona and membrane fusion entirely and is now used in a majority of IVF cycles worldwide. Embryos produced by IVF beyond those transferred are often frozen and may later be donated for research, which is how most human embryonic stem cell lines were derived. The legal frameworks governing that research differ between Canada and the United States and are the subject of lesson 12.