Fertilization

To produce a viable embryo, spermatozoa must reach the fallopian tube and adhere, fuse and release its pronuceli into the oocyte. This involves movement through the cervical passage to the fallopian tube while undergoing the acrosome reaction.

The First Obstacle: The Cervix

The cervical tract can restrict spermatozoa progression in two ways: crypts and mucus. Upon deposition, semen coagulates at the entrance due to prostate-derived alkaline and fibrinogen fluids. The alkaline quality of the gel is important to protect against the acidic environment of the vaginal passage (~pH 5.7).

The cervical mucus varies in viscosity during the menstrual cycle to prevent inappropriate progression of spermatozoa. In the days following ovulation (days 14-28 following menses) when progesterone is high, the fluid becomes more viscous to prevent any spermatozoa progression. This is one way that progesterone can be used as a contraceptive tool, by creating a permanently inhospitable environment within the cervical passage. During the estrogen only phase of menstruation (days 5-14 following menses) the mucus has high water and glycoprotein content which maximize the process of “ferning”; the mucus forms a fern-like pattern under a microscope. By analysing the mucus it is possible to determine the stage of the menstrual cycle. Although this still restricts spermatozoa progression, it is believed to help trap abnormal sperm and reducing the total number of sperm. This is a process of quality control. The high estrogen content of the cervical mucus is also believed to benefit spermatozoa movement by stimulating a “figure of 8” motif in the flagella.

The cervical wall is formed of crypts which can also prevent spermatozoa progression. During movement, spermatozoa can become trapped within crypts. The purpose of this is not clear, it may be another method to reduce spermatozoa numbers or a “reserve” as spermatozoa can survive for up to 24 hours out of the testes.

Capacitation: From Hypermotility to the Acrosome Reaction

One of the first steps for priming the spermatozoa (Figure 1A) for fertilization is capacitation, which can occur within the cervical passage or estrogen-primed uterus and oviduct. Proteolytic enzymes and high ionic strength within the secretions of the female reprodutive tract, create a suitable environment for capacitation. These conditions are not present during the progesterone high phase of the menstrual cycle.

Capacitation sees the spermatozoa becoming hypermotile (Figure 1B), losing glycoproteins that were needed for spermatogenesis, reducing cholesterol present in the plasma membrane and increasing intracellular calcium. These processes allow the sperm to become receptive to oocyte signals and prepare for the acrosome reaction; the outer acrosome membrane fuses with the plasma membrane. This leads to the expression of key membrane bound receptors for interaction and fusion with the oocyte.

Hypermotility refers to changes in flagella movement, which becomes wider in amplitude and more energetic. It is driven by the loss of membrane cholesterol (which increases membrane fluidity) and requires raised internal HCO3- to activate a HCO3- -sensitive adenylyl cyclase. Internal cAMP is increased to mediate activation of cyclic nucleotide gated channels and consequently a Ca2+ influx. The rise in intracellular calcium is via novel progesterone-sensitive cation channels present in the sperm, known as CatSper channels. These channels are believed to give directional hypermotility, guiding towards the source of progesterone production; the oocyte barrier. The directional movement of the spermatozoa is thought to be through gradients of signalling molecules released from the cumulous layer of the oocyte. This movement of spermatozoa via molecule gradients is known as chemotaxis. In this case, one such signalling molecule may be progesterone.

The cervical mucus contains pro-capacitation factors which promote the removal of cholesterol and tocopherols which were deposited in the seminal fluid. Movement through the cervical passage and uterus is driven by the spermatozoa flagella. Once the oviduct entrance is reached, spermatozoa attach to the epithelia and movement partially relies on oviduct peristalsis and cilia movement.

Spermatozoa detach from the oviduct epithelia upon reaching the oocyte. Upon ovulation the oocyte is surrounded by the cumulous barrier, composed of cumulous cells and a hyaluronic acid matrix. Hypermotility is vital for penetrating the matrix as well as expression of surface anchored hyaluronidase.

Binding and breakdown of the zona pellucida (Figure 1C) initiates the acrosome reaction (Figure 1D) and is needed for successful adhesion to the oocyte. Receptors located on the plasma membrane of the spermatozoa bind to ZP3, a component of the zona pellucida. A GPCR-mediated signal increases intracellular calcium and pH. Aggregation of GAL-Tase (β1-4 galactosyltransferase) creates multiple fusion points between the sperm plasma membrane and outer acrosomal membrane. This fusion is known as the acrosome reaction and leads to the exposure of the inner acrosomal membrane to the extracellular matrix and the release of numerous enzymes. One such enzyme is hexosaminidase B which digests GAL-T to prevent further sperm binding to the zona pellucida.

The exposure of the inner acrosomal membrane reveals new membrane proteins which can interact with additional components of the zona pellucida as well as those required for initial binding to the oocyte. This entire process must occur locally to the oocyte if fertilization is to be successful. This is because the release of hexosaminidase B and other oxidative and proteolytic enzymes (Figure 1E) rapidly make the zona pellucida impervious to sperm interaction.

Figure 1. Changes in Spermatozoa During Fertilization



Binding of Sperm and Oocyte: Formation of an Embryo

The interaction of sperm and oocyte is necessary for the ejection of the oocyte polar body and formation of the first mitotic spindle to produce a 2-cell embryo. By forming multiple adhesion points, the sperm and oocyte can fuse. There are numerous adhesion proteins implicated in oocyte-sperm binding, often identified by knock out mouse studies, that result in an infertile phenotype. The Izumo protein is present on acrosome reacted spermatozoa and knocking out the gene that codes for this protein results in infertile male mice. This protein seems to be particularly important at the point of fusion as injection of spermatozoa cytoplasm from the Izumo-null mice could still produce viable offspring. CD9 and CD81 are cell surface glycogproteins expressed on the oocyte and are classed as tetraspanin membrane proteins. They are known to be involved in complex signalling pathways and are also implicated in fertility as CD9 and CD81 knockout mice cannot produce offspring naturally.

Almost instantly following fusion, Ca2+ oscillations can be identified within the oocyte cytoplasm. The putative sperm-derived factor which drives these oscillations is phospholipase C (PLC) ζ. This was based upon evidence that Ca2+ oscillations could be induced in the oocyte by injection sperm cytosol. Alternatively, sperm-oocyte adhesion could activate PLCγ or β. The oscillations are stimulated by PLC through the cleavage of the phospholipid phosphatidylinositol (PI) to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 can then promote the release of Ca2+ from the endoplasmic reticulum by activating the IP3 receptor.

The Ca2+ oscillations are necessary to block polyspermy through the movement and release of cortical granules. These are vesicles located within the oocyte and contain factors to remove sperm-adhesion proteins from the membrane and oxidise the zona pellucida making it impermeable to additional sperm. Fusion and release of cortical granule content begins at the point closest to sperm-oocyte interaction and progressively follows round the oocyte periphery.

References

Fujinoki M. 2010, 'Suppression Of Progesterone-Enhanced Hyperactivation In Hamster Spermatozoa By Estrogen' Reproduction; 140, pp. 453-464.

 

Gupta S.K.& Bhandari B. 2011, 'Acrosome Reaction: Relevance Of Zona Pellucida Glycoproteins' Asian J Androl; 13, pp. 97-105.

 

Lee K., Wang C.& Machaty Z. 2012, 'STIM1 Is Required For Ca2+ Signaling During Mammalian Fertilization' Dev Biol; 367, pp. 154-162.

 

Mortimer S.T. 1997, 'A Critical Review Of The Physiological Importance And Analysis Of Sperm Movement In Mammals' Hum Reprod Update; 3, pp. 403-439.

 

Ramadan W.M., Kashir J., Jones C. et al. 2012, 'Oocyte Activation And Phospholipase C Zeta (Plcζ): Diagnostic And Therapeutic Implications For Assisted Reproductive Technology', Cell Commun Signal; 10 , pp. 12.

 

Sebkova N., Cerna M., Ded L. et al. 2012, 'The Slower The Better: How Sperm Capacitation And Acrosome Reaction Is Modified In The Presence Of Estrogens' Reproduction; 143, pp. 297-307.

 

Strünker T., Goodwin N., Brenker C. et al. 2011, 'The Catsper Channel Mediates Progesterone-Induced Ca2+ Influx In Human Sperm' Nature; 471, pp. 382-386.

 

Sutovsky P. 2009, 'Sperm-Egg Adhesion And Fusion In Mammals' Expert Rev Mol Med; 11, p. e11.

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