BGDA Practical 3 - Fertilization

From Embryology
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Practical 3: Oogenesis and Ovulation | Gametogenesis | Fertilization | Early Cell Division | Week 1 | Implantation | Week 2 | Extraembryonic Spaces | Gastrulation | Notochord | Week 3 | Quiz

Introduction

This page covers the process of mammalian fertilization. A complex interaction between the two haploid gametes (oocyte and spermatozoa) resulting in a single diploid cell (zygote). Following entry of the spermatazoa into the oocyte a series of changes occur within the oocyte and zona pellucida that block further fertilization by additional bound spermatozoa (polyspermy).

Due to both scientific and medical research on this process, this can now occur outside the body (in vitro fertilization) as well as fertility therapies to aid normal (in vivo fertilization). In addition, our understanding of fertilization has led to the development of a number of alternative fertility control methods.

Fertilization Dynamics

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 ‎‎Ovulation
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 ‎‎Ovulation
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 ‎‎Oocyte Transport
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 ‎‎Spermatozoa
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 ‎‎Spermatozoa Motility
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 ‎‎Fertilisation to
4 Blastomere
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 ‎‎Fertilization
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 ‎‎Fertilization
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 ‎‎Mouse Fertilisation
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 ‎‎Ectopic Polar Body
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 ‎‎Pronuclear Fusion
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 ‎‎Male Mitochondria
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Sperm Events

  1. Capacitation - removal of glycoprotein coat and seminal proteins, alteration of sperm mitochondria
  2. Binding - zona pellucida protein ZP3 acts as receptor for sperm
  3. Acrosome reaction - exyocytosis of acrosome contents (Calcium ion mediated), enzymes to digest the zona pellucida, exposes sperm surface proteins to bind ZP2
  4. Membrane fusion - between sperm and egg, allows sperm nuclei passage into egg cytoplasm

Egg Events

  1. Sperm membrane fusion - causes depolarization of egg membrane, primary block to polyspermy
  2. Cortical reaction - IP3 pathway elevates intracellular Calcium, exocytosis of cortical granules, enzyme alters ZP3 so it will no longer bind sperm plasma membrane (cortical reaction)
  3. Second meiotic division - completion of 2nd meiotic division, forms second polar body

Sperm Penetration

Click Here to play on mobile device

Human Fertilization Detail Movie Links: MP4 version | Human Fertilization Movie | Fertilization | Zygote | Movies
Approximate Timing of Early Human Events (in vitro)
Human fertilization movie 2 frame 01.jpg Human fertilization movie 2 frame 02.jpg Human fertilization movie 2 frame 03.jpg
20 min - components 28 min - spermatozoa penetrates zone pellucida 31 min - spermatozoa penetrates oocyte - fertilization
See also clock in lower righthand corner for the approximate timing of events.
Links: Human Fertilization Detail Movie | Human Fertilization Movie
Reference: PMID 22695746 J Assist Reprod Genet.
Fertilization 002 icon.jpg The animation shows:
  1. sperm moving between granulosa cells to contact the zona pellucida.
  2. sperm releasing acrosomal contents to breakdown zona pellucida.
  3. sperm fusing with egg membrane.
  4. egg releasing cortical granule contents.
  5. egg completing meiosis.

Pronuclear Fusion

Pronuclear fusion 001 icon.jpg

The animation shows:

  1. zona pellucida (yellow).
  2. polar bodies (green) which contain excess DNA.
  3. two pronuclei (blue) which loose their nuclear membranes and fuse together.
  4. chromosomes pairing to form the diplod zygote


Early zygote.jpg Human zygote two pronuclei 22.jpg
Parental genome mix 01 icon.jpg In the mouse zygote, separation of chromatin according to parental origin is preserved up to the four-cell embryo stage and then gradually disappears.

Human Zygote Size

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Enlarged Size Stage1 size with ruler.jpg

Fertilization Overview

Capacitation

  • The mammalian spermatozoa once released, must remain for a time in the female genital tract before having the capacity to fertilize the oocyte. This process involves modifying the spermatozoa.

Acrosome Reaction

  • Penetration of egg by spermatozoa is initiated by the acrosome reaction which takes different forms in different species.
  • Mammalian acrosomal lysins contain proteinases which lyse the glycoproteins of the zona pellucida.
  • The central part of the acrosome elongates into a tube which extends form the head of the spermatozoon. On contact with the egg the acrosomal membrane fuses with the sperm plasma membrane thus opening the acrosomal vesicle and liberating the granules containing acrosomal lysins.
  • The inner portion of the acrosomal membrane everts and lengthens to form the acrosomal tubule through which the sperm nucleus enters the egg.

Sperm Contact

The act of fertilization changes the egg from a stage of slow structural and metabolic decline to one of renewed activation. Morphologically egg activation is a series of surface changes immediately following sperm contact.

  • Mammals - No phenomenon comparable to the raising of the fertilization membrane is displayed. Mammalian eggs are surrounded by the zona pellucida which undergoes a structural change known as the zonal reaction after sperm penetration. On sperm contact with the egg plasma membrane, cortical granules break down as in above forms, substances liberated into the perivitelline space rapidly modify the zona pellucida resulting in a block to further sperm penetration.

Sperm Activation of Egg

  • During fertilization sperm activates the egg by induction of a calcium ion (Ca2+) oscillation within the egg's cytoplasm.
  • Induction occurs by a sperm protein factor (unidentified) which can stimulate only once calcium ion oscillations in metaphase eggs.
  • Another sperm derived factor is then responsible for the inactivation of this oscillation.
  • The activation of the egg by this calcium ion oscillation is essential for entry of the egg into the first mitotic cycle.

Zygote - Sperm Contribution

What the fertilizing sperm contributes in addition to the genetic material to the zygote differes between species.

  • Centriole - most mammalian species, sperm contribute a centriole to reconstitute the zygotic centrosome. In rodents, only a maternal centrosomal inheritance occurs.
  • Sperm Mitochondria - may enter the zygote, but are eliminated by a ubiquitin-dependent mechanism.

Perinuclear Theca - located in the sperm head perinuclear region. Contains a cytoskeletal element to maintain the shape of the sperm head and functional molecules leading to oocyte activation during fertilization.

Note - ART intracytoplasmic sperm injection techniques may introduce sperm components normally lost during in vivo fertilization.

Zona Pellucida

The specialized extracellular matrix layer lying directly around the oocyte underneath follicular cells. The structure consists of glcosaminoglycans and three main glycoproteins (ZP1, ZP2, ZP3).

After fertilization, the zona pellucida:

  1. blocks polyspermic fertilization
  2. physically protects the preimplantation embryo during early embryonic development (aided by an initial "hardening" after fertilization)
  3. aids uterine tube transport
  4. impacts upon blastocyst development

Zygote Contributions

Maternal - mitochondria, nucleolus, oocyte contributes one centriole during fertilisation.

Paternal - spermatozoon contributes one centriole during fertilisation.

Both Parents - chromosomes (inherent epigenetic differences between the paternal and maternal pronuclei), plasma membranes spermatozoon/oocyte mingle to form a mosaic plasma membrane.


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Practical 3: Oogenesis and Ovulation | Gametogenesis | Fertilization | Early Cell Division | Week 1 | Implantation | Week 2 | Extraembryonic Spaces | Gastrulation | Notochord | Week 3 | Quiz

Additional Information

Additional Information - Content shown under this heading is not part of the material covered in this class. It is provided for those students who would like to know about some concepts or current research in topics related to the current class page.
Assisted reproductive technology in Australia and New Zealand 2014

Model of gamete recognition zona pellucida

Model of gamete recognition by zona pellucida[1]


UNSW - Oocyte Biology Research Unit

Oocyte BMP15 and GDF9 effects PMID 25058588
Associate Professor Robert Gilchrist



Dr Gilchrist’s primary research interests are in the regulation of mammalian oocyte development and maturation, and the development of novel oocyte maturation techniques for infertility treatment.

UNSW Research Gateway - PubMed


Rob B Gilchrist, Alberto Maria Luciano, Dulama Richani, Haitao Zeng, Xiaoqian Wang, Michel De Vos, Satoshi Sugimura, Johan Smitz, Francois J Richard, Jeremy G Thompson Oocyte maturation and quality: role of cyclic nucleotides. Reproduction: 2016; PubMed 27422885

"The cyclic nucleotides, cAMP and cGMP, are the key molecules controlling mammalian oocyte meiosis. Their roles in oocyte biology have been at the forefront of oocyte research for decades and many of the long standing controversies in relation to the regulation of oocyte meiotic maturation are now resolved. It is now clear that the follicle prevents meiotic resumption through the actions of natriuretic peptides and cGMP inhibiting the hydrolysis of intra-oocyte cAMP and that the preovulatory gonadotrophin surge reverses these processes. The gonadotrophin surge also leads to a transient spike in cAMP in the somatic compartment of the follicle; research over the past 2 decades has conclusively demonstrated that this surge in cAMP is important for the subsequent developmental capacity of the oocyte. This is important, as oocyte in vitro maturation (IVM) systems practiced clinically do not recapitulate this cAMP surge in vitro, possibly accounting for the lower efficiency of IVM compared to clinical IVF. This review focuses in particular on this latter aspect - the role of cAMP/cGMP in the regulation of oocyte quality. We conclude that clinical practice of IVM should reflect this new understanding of the role of cyclic nucleotides, thereby creating a new generation of ART and fertility treatment options."

David Mark Robertson, Robert B Gilchrist, William Leigh Ledger, Angela Baerwald Random start or emergency IVF/in vitro maturation: a new rapid approach to fertility preservation. Womens Health (Lond Engl): 2016; PubMed 27248769

Darryl L Russell, Robert B Gilchrist, Hannah M Brown, Jeremy G Thompson Bidirectional communication between cumulus cells and the oocyte: Old hands and new players? Theriogenology: 2016; PubMed 27160446


Spermatozoa Interactions with Female Reproductive Tract

Susan S Suarez Mammalian sperm interactions with the female reproductive tract. Cell Tissue Res.: 2015; PubMed 26183721


"The mammalian female reproductive tract interacts with sperm in various ways in order to facilitate sperm migration to the egg while impeding migrations of pathogens into the tract, to keep sperm alive during the time between mating and ovulation, and to select the fittest sperm for fertilization. The two main types of interactions are physical and molecular. Physical interactions include the swimming responses of sperm to the microarchitecture of walls, to fluid flows, and to fluid viscoelasticity. When sperm encounter walls, they have a strong tendency to remain swimming along them. Sperm will also orient their swimming into gentle fluid flows. The female tract seems to use these tendencies of sperm to guide them to the site of fertilization. When sperm hyperactivate, they are better able to penetrate highly viscoelastic media, such as the cumulus matrix surrounding eggs. Molecular interactions include communications of sperm surface molecules with receptors on the epithelial lining of the tract. There is evidence that specific sperm surface molecules are required to enable sperm to pass through the uterotubal junction into the oviduct. When sperm reach the oviduct, most bind to the oviductal epithelium. This interaction holds sperm in a storage reservoir until ovulation and serves to maintain the fertilization competence of stored sperm. When sperm are released from the reservoir, they detach from and re-attach to the epithelium repeatedly while ascending to the site of fertilization. We are only beginning to understand the communications that may pass between sperm and epithelium during these interactions."

Terms

  • adplantation - Initial adhesion of blastocyst (released from zona pellucida) to uterine wall. Adplantation is followed by implantation.
  • ampulla - longest segment (approximately 2/3 of overall length) of uterine tube (oviduct or Fallopian tube). Medial segment forming the remainder of the tube is called the isthmus.
  • antrum- (L. a cave), cavity; a nearly-closed cavity or bulge. In the ovary this refers to the follicular fluid-filled space within the follicle.
  • blastocyst - the developmental stage following morula, as this stage matures, the zona pellucia is lost allowing the coceptus to adplant and then implant into the uterine wall.
  • capacitation - the process of activation of sperm, requires removal of surface glycoproteins and increased motility. The sperm now become capable of fertilizing an egg.
  • cavitates- to form a space within a solid object.
  • conceptus - the entire structure generated from the zygote.
  • fertilization - (fertilisation) The process of penetration of the oocyte (egg) by the spermatozoa and the combining of their genetic material that initiates development of the embryo. The union of two haploid gametes to form the first diploid cell, the zygote. (More? Fertilization | Spermatozoa Development | Testis Development | Ovary Development | Lecture - Fertilization)
  • fimbriae- ( L. = a fringe) fingerlike projections at the ovarian end of uterine tube.
  • follicular fluid- (or follicular fluid) the fluid found in the antrum of a secondary follicle. Secreted by cells in the wall of the follicle. This fluid is released along with the oocyte at ovulation.
  • infundibulum- funnel-shaped initial segment of uterine tube (oviduct or Fallopian tube) opening into peritoneal cavity and connected to the ampulla. The peritoneal opening sitting over the ovary.
  • morula -(L. morus = mulberry) early stage of development (12-15 cells) when conceptus is a solid ball of cells, further cell division forms the blasocyst.
  • oocyte - (egg or ovum) female germ cell.
  • ovulation- release of the oocyte from the mature follicle.
  • uterine tube- (also called oviduct or Fallopian tube) the laterally paired tubes that connect the ovary to the uterus. Is the site for oocyte fertilization and initial development of the conceptus.
  • uterine wall - the site of normal blastocyst implantation.
  • zona pellucida- glycoprotein shell that surrounds the oocyte through to blastula stage of development
  • uterus- site of embryo implantation and development. Uterine wall has 3 layers; endometrium, myometrium, and perimetrium.
  • zona pellucida- extracellular layer lying directly around the oocyte underneath follicular cells. Consists of glcosaminoglycans and glycoproteins (ZP1, ZP2, ZP3).
  • ZP1, ZP2, ZP3 - acronyms for the zona pellucida glycoproteins.
  • zygote - The first diploid cell that forms following fertilization by fusion of haploid oocyte (egg or ovum) and [S#spermatozoa|spermatozoa]] (sperm), resulting in the combination of their separate genomes. This single cell will divide by mitosis to form all cells of the embryo, fetal membranes and the embryonic component of the placenta. The term conceptus is used to describe all these cells derived from the fertilization event forming the zygote.


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Cite this page: Hill, M.A. 2017 Embryology BGDA Practical 3 - Fertilization. Retrieved September 22, 2017, from https://embryology.med.unsw.edu.au/embryology/index.php/BGDA_Practical_3_-_Fertilization

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© Dr Mark Hill 2017, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
  1. Matteo A Avella, Boris Baibakov, Jurrien Dean A single domain of the ZP2 zona pellucida protein mediates gamete recognition in mice and humans. J. Cell Biol.: 2014, 205(6);801-9 PubMed 24934154