Fertilization

Fertilization is the fusion or union of the male and female gametes - the sperm and the egg - to form a zygote. It is a union of two haploid (1N) cells to give rise to a diploid (2N) cell known as the zygote. It is the starting point for the development of an embryo, and subsequently a fully formed human. The normal site of fertilization is the ampulla of the fallopian tube (oviduct). Both the sperm and the oocyte are produced at places far away from this site of fertilization and travel across parts of the male and female reproductive tract to reach this site.

Journey of the Oocyte to the fertilization site

The journey of the oocyte to the fertilization site begins from the surface of the ovary, when a secondary oocyte (arrested in the metaphase of the second meiotic division) is released by the dominant Graafian follicle.   Once released to the exterior of the ovary, the secondary oocyte is captured by finger-like projections present at the end of the fallopian tube and called fimbria. These fimbriae gently sweep the oocyte from the ovarian surface into the fallopian tube. The secondary oocyte, surrounded by the zona pellucida and the corona radiata, travels through the fluid-filled Fallopian tube, propelled by the rhythmic contractions of the smooth muscle fibers present in the wall of the Fallopian tube assisted by beating movement of cilia of the lining epithelium of the tube. The lining epithelium of the fallopian tube contains ciliated cells and nonciliated cells. The non-ciliated cells secrete a fluid containing nutrients. The tube fluid is a vehicle that carries the ovum towards the uterus and facilitate swimming of sperms upstream in the opposite direction. The journey of the secondary oocyte is a delicate and well-orchestrated process, that ensures that the secondary oocyte reaches the site of fertilization in optimal condition. The tube ampulla is the site where fertilization typically occurs if the oocyte encounters a sperm.

Fig. 3.1.  The illustration shows the isthmus, ampulla, infundibulum of the fallopian tube, and relations of the fingerlike projections of the infundibulum (the fimbria) to the ovarian surface.

Journey of the sperms to the fertilization site

The sperms (spermatozoa) take an exceptionally long and hazardous journey from the site of their production to the site of ovum fertilization. They are produces in the seminiferous tubules of the testis and taken via a series of small ducts within the testis into the epididymis to be store and gain ability to move i.e. they become motile in the epididymis. They kept there until they are ejaculated at a high speed along the vas deferens and the urethra to the exterior of body to be deposited in the female vagina. The normal human ejaculate is about 2-4mls of semen. The semen immediately coagulates (within seconds after ejaculation). This process of ejaculation protects the sperms from the acidic environment of the vagina.

Fig. 3.2. The illustration shows the route taken by sperms from the testis (1) where they are produced into the epididymis (2) where they are stored. During ejaculation, the sperms are carried at a high speed along the vas deferens (3) towards the urethra. Secretions of the seminal vesicles (4) and the prostate (5) are added, and the thus formed semen passes along the urethra (6) to be ejaculated outside the body.

Capacitation

The sperms ejaculated into the female vagina are motile, yet they are still incapable of fertilizing the ovum; they need to be capacitated to be capable of fertilization. Capacitation is a set of physiological changes that a spermatozoon undergoes in order to be able to fertilize the ovum. It occurs within the vagina when the spermatozoa come in contact with tissues and fluids in the female genital tract. It is a vital and complex process that spermatozoa must undergo to gain the ability to fertilize an egg. Capacitation involves several biochemical and physiological changes that occur within the female reproductive tract. During capacitation, the cell membrane of sperm becomes more permeable to calcium ions, that enhance motility of the sperms motility. Moreover, capacitation involves the removal of certain proteins and cholesterol from the sperm membrane, which destabilizes the cell membrane and prepare the sperm for the acrosome reaction. Furthermore, these changes increase metabolic activities of the sperm and enhance the sperm’s ability to travel across the cervix and the uterus towards the secondary oocyte present in the Fallopian tube. Sperms cross the uterine cervix by movements of their tails utilizing energy provided by the seminal fluid  fructose and the mitochondrial sheath. The sperms cross the uterus rapidly’ within about 5-45 minutes, due to uterine muscle contractions in response to prostaglandins present in the. However, of the hundreds of millions (200-400 millions) of sperms deposited in the female vagina only few hundreds (300-400) sperms reach the site of fertilization.

Stages of fertilization

Fertilization takes place in the ampulla of the fallopian tube, usually within 24-48hr after ovulation The process of fertilization takes place in several steps or stages which involve chemical and physical events. These stages include penetration of the corona and zona, acrosomal reaction, penetration of the zona, fusion of the cell membranes, sperm head entry, cortical reaction, karyogamy, and activation of the ovum.

Penetration of the Corona Radiata

This is the first step of fertilization whereby the sperm passes in-between cells of the corona radiata. The corona radiata is single layer of follicular cells that surrounds the oocytes; and leave the ovary during ovulation along with the secondary oocyte. It is an indispensable barrier that not all spermatozoa can penetrate to reach secondary oocyte. The corona radiata and the oocytes are separated from each other and at the same time anchored together by a homogeneous extracellular material called the zona pellucida. Penetration of the zona pellucida by the sperm is accomplished by a combination of a mechanical propulsion brought about by movements of the sperm's tail coupled with digestion of a pathway through the action of acrosomal enzymes.

The Acrosomal Reaction

Acrosomal reaction is the reaction that occurs in the acrosome of the sperm as it approaches the oocyte. Optimal concentration of calcium ions and magnesium ions, along with an optimal pH are factors essential for acrosomal reactions. When the sperm head comes in contact with the zona pellucida, the outer acrosomal membrane fuses with the plasma membrane and acrosomal reaction begins. The binding of the sperm head with the zona pellucida triggers the release of the hydrolyzing enzymes contained within the acrosome including hyaluronidase and acrosin. Acrosomal reaction is a prerequisite for fertilization; only acrosome-reacted spermatozoa are able to pass through the zona pellucida and bind with the oocyte plasma membrane. Due to the acrosomal reactions, the plasma membrane of the sperm and that of the secondary oocyte get fused together and  contents of the sperm enter into the secondary oocyte. Binding of the plasma membrane of a sperm with that of the secondary oocyte depolarizes the plasma membrane of the oocyte, thus prevents entry of other sperms safeguarding against polyspermy. 

Fig. 3.3. The illustration on the left shows a secondary oocyte surrounded by the zona pellucida and cells of the corona radiata. Two spermatozoa have penetrated through the corona radiata and reached the zona pellucida; their acrosomes are in direct contact with the zona. The illustration on the right show a spermatozoon with ruptured acrosome releasing acrosomal enzymes. 

Cortical Reaction

The cortical reaction commences soon after the fusion of the plasma membranes of the sperm and the secondary oocyte. Cortical granules present under the plasma membrane of the secondary oocyte fuses with the oocyte plasma membrane and release their content of  enzymes between the zona pellucida and oocyte plasma membrane. The zona pellucida is hardened by the cortical enzymes thus hampering entry of additional sperms thus preventing polyspermy. Thus, whereas the acrosomal reaction softens the zona pellucida to allow passage of a sperm towards the oocyte, the cortical reaction hardens the zona pellucida and prevents passage of other sperms towards the secondary oocyte.  This hardened membrane impervious to sperms is sometimes referred to as the fertilization membrane.

Of the hundreds of capacitated sperms that reach the site of fertilization, only one sperm successfully gains access into the cytoplasm of the secondary oocyte. Any other sperm that tries to gain access into the oocyte is held back by the fertilization membrane formed the cortical reaction. It is a membrane formed by a change in chemistry of the oocyte cell membrane.

Fig. 3. 4. The illustration shows the steps of fertilization. A spermatozoon approaches the oocyte (1), penetrates the corona cells and comes in contact with the zona pellucida (2), undergoes acrosomal reaction (3), penetrates the zona pellucida (4), penetrates the oolemma (5), passes into the cytoplasm of the secondary oocyte and the sperm head and neck detach from the sperm tail. The sperm head enlarges to form the male pronucleus. Meanwhile, the nucleus of the secondary oocyte completes the second meiotic division and yield the nucleus of the ovum and the second polar body. The nucleus of the ovum enlarges to form the female pronucleus.

Karyogamy

The sperm as a whole cross the corona radiata and the zona pellucida, but usually it is only the sperm head and the neck (containing the proximal centriole) that pass into the cytoplasm of the secondary oocyte often leaving the middle piece and rest of the tail behind. Following the entry of the sperm head and neck into the cytoplasm, the arrested second meiotic division of the secondary oocyte is completed yielding a haploid ovum (1N) and a second polar body (1N). The nucleus within the head of the sperm enlarges and forms the male pronucleus. The proximal centriole within the sperm neck plays an important role in formation the spindle of the first cleavage division. The middle piece of the sperm, the principal piece of the tail and the second polar body degenerate and disappear. The nucleus of the ovum enlarges and becomes the female pronucleus. The male and female pronuclei lose their nuclear envelopes, and the DNA strands they contain condense to form the male and female chromosomes. Chromosomes of the haploid male pronucleus and those of the haploid female pronucleus mix up with each other and make up the diploid 2N chromosome of the fertilized ovum.  Fusion of the male and female pronuclei and mixing up of their chromosomes is called karyogamy; the thus formed fertilized ovum is called the zygote. Fertilization triggers enhancement of metabolism in the fertilized ovum, and paves the way for a number of events.

Repercussions of Fertilization

Fertilization is the starting point of a new individual’s life. It bring about a number of favorable processes and may go wrong occasionally and cause anomalies. It has several favorable consequences that include restoration of the diploid chromosome number, sex determination and initiation of cleavage. It has profound implications for the development of new life.

Restoration of diploidy

An important result of fertilization is the restoration of the diploid chromosome number, which ensures the genetic stability of the new individual and provides a solid foundation for the individual’s genetic makeup. Two haploid (1N) gametes, the sperm and the ovum, each with one set of 23 chromosomes unite to give rise to diploid (2N) zygote containing two set of chromosomes with a total of 46 chromosomes, of which 44 are autosomes and two are  sex chromosomes.

Sex Determination

Another important implication of fertilization is sex determination, which is crucial step that determines the sex of the newborn. The sperm is the determinant gamete in this process. All ova have similar chromosome compliments (22+X), whereas half of the sperms have a chromosome complement of 22+X while sperm of the other half have a chromosome compliment of 22+Y. If the ovum is fertilized by a sperm having a 22+X chromosome compliment then the chromosome complement of the zygote will be 44+XX and the newborn will be a girl. On the other hand, if the ovum is fertilized by a sperm having a 22+Y chromosome compliment then the chromosome complement of the zygote will be 44+XY and the newborn will be a girl.

Fig. 3.5. The illustration shows the process of sex determination that takes place at the time of fertilization; fertilization of the ovum with Y chromosome bearing sperms yields a boy and that with an X chromosome bearing sperm yields a girl.

Initiation of Cleavage

Fertilization also initiates cleavage, which is the first step in the complex process of development of a new individual. It is a series of rapid mitotic division. Chromosomes of the male pronucleus and those of the female pronucleus are organized by microtubules of the cell division spindle organized by the proximal centriole of the sperm neck. Within the cytoplasm of the ovum, the proximal centriole duplicates and each forms aster ray microtubules which gradually organize chromosomes of the male and female pronuclei. At the end, a complete mitotic spindle is formed with mixed up maternal and paternal chromosomes arranged along the equator of the fertilized ovum, ready for the first cleavage division.

Fig. 3.6. The illustration shows the female and female pronuclei, their fusion, and arrangement of their chromosomes by microtubules of the proximal centriole.

Anomalies of Fertilization

Fertilization is a delicate and complicated process and thus things may occasionally go wrong. This usually occur if more than one sperm gain access into the oocyte simultaneously or when a defective sperm enters into the oocyte  signaling the oocyte to form a pronucleus. The defective sperm is incapable of forming its pronucleus, accordingly only one half of the chromosomes of the thus fertilized ovum functional. When fertilization anomalies take place, the process of embryonic development is disrupted.

Polyspermy

Only one sperm fertilizes the ovum; immediately following entry of a sperm head into the oocyte a zona reaction occurs and a fertilization membrane is formed preventing entry of other sperms. The oocyte cell membrane receptors that recognize sperms are switched off and the oocyte can no longer recognize sperms, thus no more sperms are allowed in. However, in rare occasion more than sperm enter into the oocyte dead on the same time. When two sperms fertilize the oocyte, the phenomenon is called dispermy and the resultant zygote is a triploid (3N) zygote instead of the normal diploid (2N) one. More rarely three or more sperms fertilize the ovum and the phenomenon is called polyspermy. The abnormal number of chromosomes of the thus fertilized result in failure of progression of the embryonic development.

Fig. 3.7.  Of the six spermatozoa attempting to oocyte the ovum, two gained access into the oocyte and fertilized it. This dispermy and fertilized ovum contains two male pronuclei, in addition to the female pronucleus resulting in a 3N zygote.

In Vitro Fertilization

In vitro fertilization (IVF) is a widely utilized procedure for assisted reproduction to help couples that have fertility problems. This procedure involves the fertilization of a secondary oocyte in a laboratory setup, allowing the fertilized ovum until the morula stage and transfer of the morula into the uterus to carry on a natural embryonic development.  

Fig. 3.8. The illustration shows the main steps of in vitro fertilization.

Induction of Ovulation

This is the first step of in vitro fertilization (IVF) where the ovaries are stimulated by hormones to produce several secondary oocytes instead of the usual single secondary oocytes which is released from the ovary during the normal menstrual cycle. The female is injected with hormones including follicle stimulating hormone (FSH), luteinizing hormone (LH), and human chorionic gonadotropin (hCG) to trigger ovulation.

Collection of oocytes

This procedure is usually done under sedation, about 36 hours after hormonal triggering of follicular development, using transvaginal ultrasonography. Once the follicles have matured, the next step is egg retrieval. The procedure is called transvaginal ultrasound-guided fine-needle aspiration. All available mature follicles are aspirated. The aspirated fluid with the oocytes is dispensed into pre-warmed test tubes to avoid the harmful effect of temperature fluctuation that can damage the meiotic spindles of the oocytes. The aspirated follicular fluid (liquor folliculi) is then inspected promptly by an embryologist in the laboratory to retrieve the secondary oocytes and transfer them to a culture medium, for incubation.

Sperm collection

The semen sample is collected from the male partner on the same day of oocyte retrieval. The semen is collected in a sterile, nontoxic jar.  The sperm are then processed and prepared in the laboratory to select the healthiest and most motile sperm for fertilization. After the initial assessment is completed, the sperm sample is washed, the seminal fluid is removed, dead and weak sperms are motile sperm are removed, and motile sperm are collected. Washing the sperms capacitates them and increases their capacity of fertilizing.

In vitro fertilization

The selected motile healthy sperms are combined are added to the oocytes kept in the culture medium for normal fertilization to take place. In about 18 hours it is possible to determine if fertilization has taken place.  After making sure that fertilization has occurred, the zygote is transferred to an appropriate culture medium that resembles Fallopian tube fluid and provides an appropriate environment for cleavage and subsequent embryonic development. The developing embryos are closely monitored for a few days to ensure appropriate quality. It takes about 2 to 4 days to establish whether the embryos are developing properly. The embryos often stay in the laboratory for a total of five days. The best quality embryos are then selected for transfer to the uterus.

Embryo Transfer

Embryo transfer is the final step of the IVF procedure. A thin catheter is used for the procedure. The selected embryos are loaded into the catheter and gently placed into the uterus via the vagina. This procedure is relatively simpler than the oocyte collection and does not require anesthesia or sedation. In about 1-5 days after transfer, one or more of transferred embryos get implanted into the uterine endometrium and the embryonic development proceeds normally thereafter. 

Approximately two weeks after embryo transfer, a pregnancy test is conducted to determine if implantation and pregnancy have occurred. The success rate of IVF varies depending on several factors including age of the woman and sperm viability but generally rages between 40% and 55%. IVF increases the likelihood of multiple pregnancies, such as twins or triplets.

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