FERTILIZATIONDR (MRS) V B BUCKTOWAR

LECTURER, OBG DEPT.

DR. D Y PATIL MEDICAL COLLEGE

MAURITIUS

LEARNING OBJECTIVES

Knowing the necessary steps which lead to spermatozoa being ready

Describing how the enabling of the spermatozoa takes place

Describing how the spermatozoon penetrates into the oocyte

Knowing the process whereby a zygote is formed

Cleavage

Blastocyst formation

We have learned that while the ovarian follicle is growing, the oogonium within it undergoes maturation

Oogonium enlarges to form a primary oocyte.

The primary oocyte undergoes the first meiotic division to shed off the 1st polar body and becomes secondary oocyte.

At ovulation, the second meiotic division is in progress and a spindle has formed for separation of the second polar body.

At this stage the ovum enters the infundibulum of the uterine tube and passes into the ampulla.

Stages of follicular development

Fertilization - the action or process of fertilizing an egg, involving the fusion of male and female gametes to form a zygote.

Complex sequence of cordinated molecular events that begins with contact between a sperm and an oocyte and ends with the intermingling of maternal and paternal chromosomes .

For successful fertilization, a mature spermatozoon (sperm cell) must penetrate into the mature oocyte.

At the same time this is also an encounter between one of the smallest human cells with one of the largest:

The head of the sperm has a diameter of only 4mm, whereas the egg cell has one of 20mm

Fertilization of the ovum occurs in the ampulla of the fallopian tube.

One spermatozoon pierces the zona pellucida and enters.

The fertilization process takes approximately 24 hours.

One can speak of a successful fertilization when the genetic material of the male spermatozoon has united with that of the female oocyte thereby creating a new, unique individual.

An Oocyte(2) following ovulation, surrounded by a cloud of cumulus cells(1), the corona radiata, which form a cloak around it.

Spermatozoa in a native ejaculate. These must still mature in several steps during their way through the female genital tract before they are able to fertilize the oocyte.

Delving deeper

When the spermatozoon leaves the testis it is not yet capable of fertilizing an oocyte. What changes must it undergo to make it capable of uniting with the oocyte (capacitation)?

How does the oocyte get from the ovary into the fallopian tube?

What makes it possible for the spermatozoon to penetrate into the oocyte?

How is it made sure that a spermatozoon from another species is unable to deposit its genetic material?

When the fertilization is completed, the genes of the two parents have united. How does this union of the two genomes take place?

The female genital tract1 Ovary2 Infundibulum3 Fimbriae4 Fallopian or uterine tube5 Ampullary part of the tube6 Uterine musculature7 Uterine mucosa8 Cervix9 Portio10 Vagina11 Ligamentum ovarii proprium12 Suspensory ligament of the ovary13 Ovary cut open (follicles in various stages)

Approximately one-and-a-half days before the midpoint of the cycle, the concentration of the luteinizing hormone (LH) rises steeply.

The LH peak is responsible for a whole series of processes: triggering ovulation.

With the LH peak the following maturation steps are now triggered in and around the oocyte - up to ovulation:

In the oocyte:

Termination of the first meiosis with ejection of the first polar bodyBegin of the second meiosis with arrest in the metaphaseMaturation of the oocyte cytoplasma by preparing molecules and structures that will be needed at the time of fertilization.

The results of these processes are:

 The correct placement of the uterine tube infundibulum upon the ovarian surfaceThe rupture of the follicle wall and the flow of the follicle fluid with the oocyte into the infundibulumThe inhibition of the maturation of further follicles

The acquisition of the oocyte by the fallopian tube

The fimbriae of the tube place themselves around the ovarian stigma and seal this location off.

Via a rotation about the axis of the suspensory ligament of the ovary and the ovarian ligament the ovary can turn the follicle that is about to rupture towards the fallopian tube.

When the surface ruptures the mass of cumulus cells, which are saturated with hyaluronic acid and which shelter the oocyte, reach the fallopian tube together with serous yellow follicle contents.

1 Ovary2 Follicle that is about to rupture3 Infundibulum4 Fimbriae

5 Fallopian tube6 Ligamentum ovarium proprium7 Suspensory ligament of the ovary

1 Fallopian tube cut open with the tube mucosa that lies in folds

2 Closely apposed fimbriae3 Follicle fluid that has flowed out

4 Secondary oocyte with corona radiata

5 Ovary with follicles in various stages of development and atresia

6 Pellucid zone

7 First polar body

8 Secondary oocyte

9 Cells of the corona radiata

10 Arrested spindle apparatus

OOCYTE ACQUISITION MECHANISM

The oocyte in the cloud of cumulus cells following ovulation In the fallopian tube, the secondary oocyte is surrounded by the corona radiata and scattered parts of cumulus cells (so-called cumulus cell cloud).

The fluid that lies in between is sticky and stringy (effect of the hyaluronic acid) with a high concentration of progesterone (to attract the spermatozoa).

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Secondary oocyte (in arrestedmetaphase of the 2nd meiosis)Corona radiataFollicle fluidScattered groups of cumulus cells

The oocyte now "waits" in the fallopian tube on fertilization by the sperm.

The matrix of hyaluronic acid holds it "captive" there, so to speak.

After a number of hours the matrix liquefies more and more

and the oocyte is gradually transported towards the uterus by the ciliary beats of the tube's epithelium cells.

Spermatozoa maturation steps The spermatozoa go through several temporal maturation steps in a

series of different locations in order to be capable of penetrating into the oocyte.

Oocyte's maturation steps involve the storing of yolk and the process of meiosis,

FUNCTIONAL MATURATION STEPS are required with the spermatozoa, which involve their motile abilities & ability to penetrate through the egg covering.

The spermatozoa experience an initial maturation step during the time they are "stored" in the epididymis.

When the ejaculation occurs, a second step follows that leads to a sudden activation of their motility.

Spermatozoa maturation steps-summary

The third step takes place during their stay in the female genital tract, especially during the ascension towards the ovary through the uterus and fallopian tube.

The spermatozoa experience thereby the so-called capacitation.

Finally, the last activation step follows: the acrosome reaction in the immediate vicinity of the oocyte.

The maturation and activation of the spermatozoa occur in the following four steps:

Storage in the epididymis -Maturation

Ejaculation -Activation

Ascension to the ovary -Capacitation

Near the oocyte -Acrosome reaction

Only a spermatozoon that has undergone an acrosome reaction is capable of binding to the pellucid zone of the oocyte.

Maturation steps of the spermatozoa in the epididymis: Through the deposition of new proteins in the nucleus, the DNA becomes more

condensed.

The sperm head becomes smaller, thereby, and more compact.

The cytoplasma is further reduced, making the sperm cells more slender.

The ability for motility is achieved.

The structure of the plasma membrane is altered. This has effects on the motility, the capacitation ability and the ability for the acrosome reaction.

Only spermatozoa that have passed through the epididymis are mature enough to be capable of motility.

While the spermatozoa are pushed through the deferent duct and the urethra, a large volume of secretions of various glands are mixed in.

This fluid part of the ejaculate is known as the seminal plasma.

The path of the sperm cells to the oocyte - capacitation Following the ejaculation a large number of sperm cells are deposited in vagina,

near the uterine cervix (portio vaginalis uteri).

The path that the sperm cells must travel from the portio to their meeting with the oocyte in the ampullary part of the fallopian tube is 13-15 cm long.

Along this stretch, sperm cells go through a further maturation process, the so-called capacitation.

Of the roughly 200 million ejaculated sperm cells only a few hundred are able to traverse the long way through the cervix, the uterus, and past the fallopian tube isthmus to the tube's ampullary region to there meet oocyte.

The cervical canal

At the time of ovulation the properties of the cervix mucus changes from a "sperm-hostile "environment to a very "sperm-friendly" one.

Before the ovulation (Fig. 26) the cervical canal is narrow and the cervix mucus is strongly meshed (it forms the so-called cervical barrier) that hinders the passage of sperm cells.

At the time of ovulation (Fig. 27) the cervix wall becomes looser and the canal wide.

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Sperm cellsMucus fibers (strongly meshed)Crypt of a cervix gland

Capacitation is what one calls the changes that lead to hyperactivity of the spermatozoon and which later allow the spermatozoon to go through the acrosome reaction.

7 hours

Much of it in uterine tube

Only capacitated sperm can pass through corona cells and undergo acrosome reaction

Penetrating the cumulus cells

Enzymes are released by the acrosome reaction; the hyaluronidase dissolves the intercellular matrix between the cumulus cells, other enzymes dissolve the pellucid zone that lies around the oocyte.

Normally, the acrosome reaction of the spermatozoa takes place first when they encounter the pellucid zone.

Upon arriving at the pellucid zone, these sperm cells themselves undergo an acrosome reaction and a further amount of hyaluronidase and other enzymes are released.

In this way, the cumulus cells is further loosened up and more and more sperm cells obtain the possibility of undergoing the acrosome reaction themselves at the pellucid zone.

The hyperactivity of the spermatozoa caused by the capacitation is a decisive factor to go through the mass of cumulus cells.

The acrosome reaction

During the acrosome reaction the contents of the acrosome are released outwardly.

The cell membrane of the spermatozoon fuses with the outer membrane of the acrosome.

The contents of the acrosome flow out through the resulting pores.

After binding to zona pellucida

Induced by zona proteins

Culminates release of enzymes needed to penetrate the zona pellucida including acrosin and trypsin-like substance

RESULT: The penetration of the pellucid zone

Fertilization

Phase-1: penetration of corona radiata

Phase-2: penetration of zona pellucida

Phase-3: fusion of oocyte and sperm cell membrane

Phase-1: penetration of corona radiata

200-300 million spermatozoa deposited in female genital tract

300-500 reach the site of fertilization

Only capacitated sperms pass through corona

Phase-2:penetration of zona pellucida

Zona pellucida: Glycoprotein shell surrounding egg

•Maintains sperm binding and induces acrosome reaction

•Acrosin allows sperm to penetrate zona

Phase-3: fusion of oocyte and sperm cell membrane

•Fusion between plasma

membrane of oocyte and sperm

•Head & tail of spermatozoon enter cytoplasm of oocyte

•Plasma membrane left on oocyte surface

Process of Fertilization

Phases of fertilization

Completion of the second meiotic division of the secondary oocyte and formation of female pronucleus.

Phases of fertilization

Formation of the male pronucleus

The condensed DNA must be unpacked as a first step, i.e., decondensed.

A nucleic membrane encloses the decondensing DNA.

The formation of a (pro)nucleus is necessary for the subsequent synthesis phase in which the DNA is duplicated.

Oocyte containg two haploid pronuclei is called as Ootid

The formation of the maternal pronucleus

At the same time that the paternal pronucleus forms, the maternal pronucleus is also generated. The penetration of the spermatozoon into the secondary oocyte causes the resumption and termination of its second meiosis.

After the 2nd polar body is created the maternal chromosomes that remain in the oocyte are enclosed by a nucleic membrane and decondensed.

Synthesis phase of the DNA in the pronuclei

The DNA must be duplicated before each commencing cell division so it can be distributed among the daughter cells.

In an impregnated oocyte this happens for every chromosome set – the one from the father and the one from the mother – each in a separate pronucleus.

The time needed for the duplication of the DNA amounts to roughly 12-18 hours. During this time span, the two pronuclei also get closer to each other spatially.

Phases of fertilization- Approach of the pronuclei

The paternal and the maternal pronuclei move towards each other with the help of microtubules, which begin to be formed immediately after penetration of the spermatozoon.

They grow in a star-like pattern out of the paternal centrosome directly beside the forming paternal pronucleus (= formation of an aster made of dozens of microtubules).

The microtubular proteins themselves arise from the cytoplasma of the oocyte.

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Paternal pronucleusMaternal pronucleusPaternal centrosome"Inner bodies"Maternal astral microtubule

Phases of fertilization- Approach of the pronuclei-contd.

the microtubules of the aster pull the pronuclei together in the center of the oocyte.

Simultaneously , the synthesis of the DNA is taking place in the pronuclei.

This duplication takes roughly 12 hours. The pronuclei grow in size in this time.

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Paternal pronucleusMaternal pronucleusDuplicated paternal centrosome"Inner bodies"

The formation of the zygote

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Nucleic membranes of thepronuclei, as they are dissolvingMicrotubules of the mitotic spindle

After the two pronuclei have come close together the nucleic membranes of both pronuclei dissolve and the chromosomes of both align themselves on the spindle apparatus at the equator.

The zygote, the first cell of a new organism with an individual genome (2n4C) is created by the alignment of the maternal chromosomes together with the paternal ones on a common spindle apparatus.

The formation of the zygote-contd.

The mitotic spindle divides the chromosomes that have just been brought together into the two first cells of the embryo.

This proceeding towards the two-cell stage occurs on average between 22 and 26 hours after fertilization.

The goal of the fertilization cascade is thus achieved:

The fabrication of a diploid set of chromosomes

The determination of the chromosomal gender of the new individual

The induction of normal "cleavage division" for embryogenesis.

Bi-parental inheritance

The zygote - by definition the first cell of the embryo

39Results of fertilization

Restoration of diploid chromosomal number

Bi-parental inheritance

Primary sex determination

Initiation of the cleavage of the zygote

40Cleavage

Two celled zygote Mitotic divisions Cells are called blastomeres Clevage normally occurs as the zygote passes along the uterine tube

towards the uterus Morula

Inner cell mass – embryo proper Outer cell mass – Trophoblast - Placenta

Cleavage

The cleavage divisions up to the morula stage

Approximately 24 hours after fertilization the impregnated oocyte begins with the first cleavage division.

The morula, a collection of around 30 cells (blastomere), is created at about 96 hours.

Because these cells arise only through the cleavage of the zygote and all are found inside the pellucid zone, which cannot expand, no growth is seen.

Every new cell is thus only half as large as the cell from which it derives.

The name of this stage comes from its resemblance to a mulberry, since it really looks like a collection of spherical cells.

How a blastocyst is engendered On the 4th day after insemination the outermost cells of the morula are still enclosed within the pellucid zone.

These begin to join up with each other (so-called compaction). An epithelial cellular layer forms, thicker towards the outside, and its cells flatten out and become smaller.

A cavity forms in the interior of the blastocyst into which fluid flows (the so-called blastocyst cavity).

The two to four innermost cells of the morula develop into the so-called inner cell mass of the blastocyst.

The actual embryo will develop solely from these cells (embryoblast).

These cells are concentrated at one pole, the embryonic pole of the blastocyst.

Thus,an outer cell mass (the trophoblast), consisting of many flat cells, and the embryoblast, formed from just a few rounded cells.

From the trophoblast the infantile part of the placenta and the fetal membranes will arise.

1 Embryoblast 2 Pellucid zone 3 Trophoblast 4 Blastocyst cavity

45Following Blastocyst formation

Zona pellucida disappears. End of first week of development

Beginning of the Implantation

(Penetration of the uterine wall)

8 cell stage

Fertilizationampulla

Male & female

pronucleus

2 cell stage

morula blastocyst

1) cleavage and blastocyst formationcleavage: mitosis of the zygote---blastomere: cells from cleavage 30h: 2 cells 40h: 4 cells 72h: 12-16 cells – morula formation

Thank you

References: www.embryology.ch

www.youtube.com/watch?v=BFrVmDgh4v4