Fertilisation

Introduction

Fertilisation is an internal process in mammals, the gamete cells however are not fully mature when they leave the gonad (either testicle or ovary) and so continue a process of maturation right up to actual fertilisation, the ovum matures fully when it undergoes its second meiotic division, the process for sperm is as follows:

• • Sperm leaving the Sertoli cell are non-motile and their DNA (located in the ‘head’ of the sperm) is not condensed, sperm are not fertile in this state.
  • It normally takes 8-15 days for sperm to mature as they pass through the epididymis
  • Over this period, certain changes occur in the membrane and nucleus of the sperm. These changes are dependent on androgens.
  • The membranes of the sperm also develop zona pellucida constituent receptors (ZP3 protein) which is important for fertilisation

Ejaculation

Upon ejaculation sperm are deposited either in the anterior vagina or directly into the uterus. Despite billions of sperm being deposited only a few (100-1,000) sperm are considered competent (able to reach the site of fertilisation – the infundibulum of the oviduct, around 8-10 hours after ejaculation). The majority of these sperm pass into the peritoneum.

Up to 95% of sperm are expelled out of the vagina to be destroyed by macrophages. Oestradiol increases uterine activity to help passage sperm through the cervix and along the uterus. Only a small amount of sperm makes it to the isthmus, which acts in some species as a reservoir for the sperm for 24-40 hours after coitus. The sperm reach the isthmus by combination of the uterine activity and their own motility, taking around 2-7 hours. Abnormal sperm are prevented from passing any further up the female reproductive tract.

Survival times for the gametes in the female reproductive tract:

Ionic constituents such as citric acid in the isthmus inhibit sperm. This makes the sperm less motile. However at ovulation waves of sperm are periodically released into the ampulla, this is thought to be under the influence of hormonal control. At this point sperm become hyperactivated; they show exaggerated movement as they move to meet the egg influenced by a possible chemoattractant.

The fimbria (finger like projections at the end of the fallopian tube) help to move the ovum down the oviduct. Exogenous steroids (such as prescribed drugs) can affect the passage of the oocyte that is the basis of the mechanisms for the morning after pill in humans and misalliance steroid treatment in bitches.

Sperm Capacitation

Capacitated spermatozoa are ones that have undergone changes in the female reproductive tract enabling them to fertilise the ovum, this occurs in the isthmus. This process is reversible and no morphological changes occur. These changes can also be induced by simple dilation in a solution.

Capacitation occurs when there are alterations made to the plasma membrane. These include changes in the charge and the removal of cholesterol that decreases the cholesterol: phospholipids ratio.

Capacitation is responsible for the hyperactivated motility pattern, which leads to the wider and stronger beats of the tail to enhance motility.

The Acrosome Reaction

The sperm must penetrate through two layers of the ovum to fertilise it, these include the cumulus cells with their extracellular matrix and the zona pellucida. Sperm motility is important here to allow them to wriggle through the cumulus layer to reach the zona pellucida. The zona layer consists of 3 glycoproteins – ZP1, ZP2 and ZP3. ZP1 is mainly structural but ZP3 is a Ligand for the attachment of sperm and is species specific. Sperm develop a receptor for ZP3 as they mature in the male seminiferous tubules. The attachment of the sperm ZP3 receptor to the zona Ligand is responsible for the triggering of the acrosome reaction (in capacitated sperm). It is possible for non-capacitated sperm to attach but the acrosome reaction will not be induced.

Stimulation of the acrosome reaction requires an increase in intracellular Ca²⁺ that is induced by an ionophore – promoting rapid transport of Ca across the plasma membrane into the cell. The acrosome of the sperm cell swells and the membrane fuses with the over-lying plasma membrane. Vesicles are formed, which is followed by the removal of the outer membrane. Intracellular Ca and cyclic AMP levels increase.

After the acrosome reaction, binding of ZP2 receptors on the now exposed inner acrosome layer is essential to hold the oocyte and sperm together. The sperm is then able to work its way through the zona layer, with the aid of a digestive enzyme acrosin as well as its motility.

Summary:

• • Sperm reaches oocyte and releases hyaluronidase to digest hyaluronic acid rich cumulus cells
  • The enzyme acrosin is able to digest zona layers and membranes of the oocyte
  • Sperm cell membrane fuses with the egg cell membrane
  • Contents of the acrosome head are released into the egg
  • ZP3 ligand on the ovum binds to ZP3 receptor on the sperm
  • Binding of the ZP3 components releases further enzymes which allow the sperm to fuse with the egg

Sperm and Egg Fusion

Once sperm have made it through the zona layers and into the peri-vitalline space, the sperm head aligns away from the oocyte DNA to avoid complications arising. Sperm movement ceases and microvilli on the surface of the ovum interact with the sperm. This results in a change in electrical charge on the vitalline membrane resulting in hyperpolarisation.

Zygote Activity

The next steps of fertilisation ensure a diploid nature of the forming embryo and the prevention of polyspermy or meiosis.

Immediately after sperm and egg fusion intracellular Ca levels rise dramatically. This causes cortical granules to release their contents into the peri-vitalline space that disrupts the ZP receptors and prevents further binding by any other sperm. The increased Ca also activates secondary oocyte meiotic division.

Sperm head proteins decondense and a few hours after fusion, membranes form around each set of gamete haploid DNA to form two pronuclei. The pronuclei approach each other, the nuclear envelopes disappear and the two chromosome sets aggregate in prophase – this completes the process of fertilisation as the zygote can now begin mitotic division.

The majority of zygote cytoplasm comes from the oocyte and so even after vesicle membrane breakdown oocyte DNA dominates. Successful fertilisation requires the correct formation of proteins from the oocyte, most of which would have formed when the oocyte was still in the follicle – correct follicular growth is therefore important for successful fertilisation.

Mitosis of the zygote continues – cell cleavage results in the formation of blastomeres that are large cells. When the zygote is 8-cells large, the embryonic genome becomes active and it begins to synthesise its own ribosomal RNA. It is believed that cell differentiation occurs at the 8 cell stage as well, because separation of the cells at the 4 cell stage will result in 4 identical offspring, however separation at the 8 cell stage only results in a maximum of 5 identical offspring.

The zygote arrives in the uterus in the morulla or blastocyst stage still in the zona.

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