FERTILIZATION AND THE FIRST WEEK OF LIFE

Professor Alfred Cuschieri

Department of Anatomy

University of Malta

Objectives

By the end of this session the student should be able to:

o Name the mechanisms and barriers involved in sperm transport along the female genital tract

o State the events occurring in the spermatozoa immediately prior to fertilization

o State the events occurring in the oocyte immediately after sperm penetration

o Explain why mitochondrial inheritance is usually transmitted by females but manifests itself in both sexes.

o Define the terms morula, blastomeres, blastocyst, embryoblast and trophoblast

o Outline the procedures involved in in-vitro fertilization

o Name other types of assisted conception

o Outline the meaning of human cloning

o Outline the importance of transgenic animals in medical research

The great event of fertilisation occurs in the outer one-third of the uterine tube.

Sperm Transport in the Female Genital Tract

o occurs by a combination of two mechanisms :

– Motility of spermatozoa – they move at the speed of 2-3 mm/hour

– Contractions in the female genital tract

–

o The greatest barrier to sperm transport is the mucus plug in the cervix

– Before ovulation it is a fluid E-type mucus

– After ovulation it is a viscous G-type mucus difficult to penetrate

o May last 2 to 3 days before reaching the ampulla

o Less than 1% of spermatozoa reach the ampulla

Spermatozoa released from the seminiferous tubules and oocytes released from Graafian follicles are still not functionally mature. They have to undergo further changes before fertilization can be completed.

The events that occur in the spermatozoa prior to fertilization are:

1. Sperm maturation

a. Occurs in the male genital tract:

- in the epididymis is stimulated by epididymal proteins

- in the urethra by mixture with secretions of the seminal vesicle, prostate and bulbo-urethral glands

b. Is associated with changes in cell surface glycoprotein

c. Results in:

- the acquisition of progressive motility

- the ability to fertilize an oocyte

2. Sperm Capacitation

a. Occurs in the female genital tract

b. Is stimulated by secretions in the vagina , uterus, and uterine tubes

c. Results in capacity to pass through the corona radiata

d. Involves removal of surface coatings and changes in plasma membrane

e. Can be produced in vitro by washing with salt solution

3. The acrosome reaction

a. Is stimulated by sperm-binding glycoprotein molecules in the zona pellucida (ZP3, ZP1 and ZP2 ligands)

b. Is accompanied by Ca²⁺ influx into sperm

c. Results in the release of acrosomal enzymes, which include hydrolytic enzymes and

d. Involves fusion of the acrosome membrane and plasma membrane

e. Is necessary for sperm penetration through the zona pellucida

A spermatozoon has to penetrate four layers before it fertilizes the oocyte:

Several spermatozoa bind with the zona pellucida. The sperm binding molecules (ZP3 ligand) in the zona pellcida stimulate the acrosome reaction.

Three changes occur in the oocyte after penetration of vitelline membrane:

Fertilization has two important genetic consequences:
1. The diploid chromosome number is restored (2n)
2. The genetic sex of the zygote is determined (XX or XY)

The spermatozoa determine the sex of the embryo. There are X - bearing spermatozoa and Y-bearing spermatozoa. The oocyte always contributes an X chromosome.

The cytoplasmic organelles of the zygote are almost entirely maternal

• Mitochondrial DNA is almost entirely maternal

• Mitochondrial genetic diseases are generally inherited through the mother but may affect both sons and daughters

•The genes in mitochondrial DNA code for enzymes required for oxidative phosphorylation

• Most mitochondrial diseases affect muscle and nerve

• Examples of mitochondrial inheritance are:

– mitochondrial myopathy (affects muscle)

– Leber’s optic atrophy (affects optic nerve)

Fertilization is a process that extends over a period of 4 to 6 hours. It commences on contact of a spermatozoon with an oocyte membranes and ends with the fusion of male and female pronuclei.

Cleavage

- Immediately after sperm penetration the zygote contains male and female pronuclei.

- The zygote immediately proceeds to the first mitotic division in the plane of the first polar body.

- Cytokinesis results in two blastomeres enclosed within the zona pellucida.

- The second mitotic division occurs at right angles to the first and results in four cells

- The mass of cells is termed a morula; the component cells are termed blastomeres.

- The morula does not increase in size and remains bound within the zona pellucida.

- The cells become progressively smaller as their number increases.

- At day 4 the morula contains about 32 blastomeres

The morula is differentiated into two groups of cells:

1. A small group of internal blastomeres called the inner cell mass or embryoblast, which gives rise to the embryo

2. The surrounding cells, termed the outer cell mass or trophoblast, which give rise to the placenta and membranes

On the 4^th day the morula is transformed into a blastocyst. In this process:

- the zona pellucida ruptures

- fluid accumulates between the blastomeres forming a blastocyst cavity

- the embryoblast is situated at one pole of the blastocyst, the embryonic pole

In-vitro Fertilization involves the following main steps

1. Ovulation is induced in the mother by human menopausal gonadotrophin (HMG) - FSH

2. Multiple oocytes are collected by laparoscopy

3. Meanwhile spermatozoa are collected and are capacitated

4. Oocytes are exposed to the capacitated spermatozoa in a culture dish

5. Fertilization is observed microscopically

6. The conceptus is grown in culture to the blastocyst stage

7. The conceptus is implanted in the uterus on the fourth day

Other forms of assisted fertilization include:

- IVF with donated gametes - donated spermatozoa, oocytes or both are used for IVF. When the oocytes are donated the zygote is implanted in the uterus of a surrogate mother

- GIFT - Gamete Intra-Fallopian Transfer. This is a modified form of artificial insemination where the oocytes and spermatozoa are placed in the uterine tube so that fertilization can occur naturally

- ZIFT - Zygote Intra-Fallopian Transfer – Newly fertilized zygotes are transferred immediately into the fallopian tubes so that they develop within a natural environment, while they are transported by ciliary action to the uterus

- ICSI - Intra-cytoplasmic Sperm Injection – A spermatozoon is picked up using a micropipette, which is then used to puncture the zona pellucida and vitelline membrane and the sperm is injected into the oocyte cytoplasm. This method has become very popular and can be used when sperm count and sperm motility are very low.

Assisted fertilization is now widely used in cases of infertility. It has several ethical implications.

Cloning is the production of identical copies of cells. In popular usage it refers to cloning of zygotes. Clones have identical copies of DNA. The following diagram outlines the main steps in the classical technique of cloning. It requires a zygote whose nucleus is then replaced by a donor nucleus.

Cloning was performed over 40 years ago in frogs. The technique of cloning was successfully applied to mammals in 1984, with the cloning of the sheep, Dolly. In recent years it was performed in several mammals, but the success rate is still very low. The first human clone was reported in December 2002, but the report has not yet been scientifically validated.

Alternative methods of cloning:

Unfertilised oocytes can be used for cloning. The haploid nucleus of the oocyte is replaced by a donor nucleus. The zygote is then induced to begin dividing

It is also possible to induce an unfertilised oocyte to develop into a zygote directly by electrical stimulation. Fusion of the two nuclei produced during the first meiotic division results in a diploid nucleus containing DNA identical to its mother’s. This process is termed parthenogenesis.

Transgenic animals are widely used in research for studying the effects produced by mutant human genes. Using transgenic animals the molecular, biochemical, and morphological changes produced by the mutant genes can be analysed. They are also used for studying the effects of medicinal drugs for the therapy of genetic diseases.

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