Book Volume 1
Page: i-i (1)
Author: Ryuzo Yanagimachi
Page: ii-iii (2)
Author: Elisabetta Tosti and Raffaele Boni
Page: iv-vii (4)
Author: Elisabetta Tosti and Raffaele Boni
Page: viii-viii (1)
Author: Elisabetta Tosti and Raffaele Boni
Page: ix-x (2)
Author: Elisabetta Tosti and Raffaele Boni
Electron Microscopy of Mammalian Oocyte Development, Maturation and Fertilization
Page: 1-37 (37)
Author: Poul Hyttel
The ultrastructure of the oocyte and zygote reveals in great details the processes of oocyte growth, maturation and fertilization. In this Chapter these details are addressed in cattle in comparison with pig, horse, fox, mouse and man. In the growing oocyte a variety of common and oocyte-specific organelles and inclusions are build up resulting in the complex ultrastructure of the oocyte; one of the largest cells of the mammalian body. During development, the oocyte is surrounded by cumulus cells of which the innermost establish gap junctions with the oolemma. However, when oocyte maturation is initiated and meiosis resumed, this intimate contact is broken and the cytoplasm of the oocyte is restructured towards a more independent fate allowing for cytoplasmic oocyte maturation. As one important aspect, cortical granules migrate to solitary positions along the oolemma immediately prior to ovulation. The fertilizing spermatozoon completes acrosome reaction on the surface of the zona pellucida, penetrates the zona, and fuses with the oolemma at the equatorial segment. Consequently, the oocyte is activated resulting in exocytosis of the cortical granules, establishing the block against polyspermic fertilization, and in resumption of meiosis from metaphase II. The maternal and paternal chromatin is gradually surrounded by nuclear envelope developed from the smooth endoplasmic reticulum to form pronuclei that later swell to their typical spherical shape. Later, the pronuclei appose each other, the nuclear envelopes are dissolved, and the maternal and paternal chromosomes are arranged in the center of the zygote forming the metaphase of the first mitosis.
Intra- and Intercellular Molecular Mechanisms in Regulation of Meiosis in Murid Rodents
Page: 38-63 (26)
Author: Alex Tsafriri and Nava Dekel
Overviewed is the life history of murid female germ cells from their first appearance as primordial germ cells during embryogenesis, their migration, proliferation and colonization of the genital ridge, the prospective gonad; the differentiation of primordial germ cells into oocytes that embark on the protracted meiosis in the embryo; the arrest of the meiosis in the newborn; the resumption of meiosis in the sexually mature animal, and finally the release of fertilizable ovum at ovulation. Emphasized are recent advances in the molecular regulation of meiosis initiation in the embryo and its resumption in the mature follicles in response to the ovulatory stimulus by luteinizing hormone.
Advances in the understanding of the regulation of meiotic resumption are presented in their historical perspective and the development of appropriate experimental models. Within the context of somatic cell regulation of meiosis resumption, the apparent paradoxical involvement of 3’5’ cyclic adenosine monophosphate in both inhibition and stimulation of meiosis are discussed; the role of somatic cell-oocyte gap junctions and the recently established obligatory role of follicular epidermal growth factor-like molecules in the mediation of the gonadotropic stimulation of meiosis and ovulation.
The involvement of steroids in mediating the gonadotropic stimulation of meiotic resumption in fishes and amphibians is well established. Nevertheless, the available data do not seem to support an obligatory role of steroids in meiotic resumption in mammals, and, particularly, not in murids. Apparently the evolution of hierarchical follicle growth and the consequent high intraovarian steroid levels resulted in the abandonment of steroids as a reliable signal for meiosis in mammals.
The Enhancers of Oocyte Competence
Page: 64-70 (7)
Author: Yves JR. Menezo and Kay Elder
During oocyte maturation, nuclear maturation i.e. the condensation of chromosomes and formation of the meiotic apparatus is generally considered as the most significant physiological process. However, disruption of the contact between granulosa cells and the oocyte leads to spontaneous nuclear maturation of oocytes which, however, have poor or nil developmental competence after fertilisation. Acquisition of cytoplasmic competence, i.e., the ability to sustain early development of embryos with high developmental potential, is the result of concomitant synergic actions of gonadotrophins and growth factors. The transforming growth factor beta superfamiliy, the epidermal growth factor network, insulin growth factors and growth hormone together with Leukaemia inhibiting factor are partners in the mechanism of acquisition of developmental competence in oocytes. These interactions allow the (quantitatively and qualitatively) correct storage of mRNAs and proteins necessary for the early embryonic divisions prior to genomic activation. However, the quality of the endogenous pool of metabolic intermediates such as (sulphur) amino acids is a mandatory prerequisite for oocyte activation, sperm decondensation and further on early embryo divisions. A correct timing of translation of the mRNAs stored during oocyte maturation is mandatory for the successful passage of the maternal to zygotic transition, usually considered as the critical step in early embryonic developmental arrest.
Genomic Regulation through RNA in Oocyte Maturation of Large Mammals
Page: 71-79 (9)
Author: Marc-Andre Sirard*
The oocyte is a unique cell amongst the 212 cell types that makes an individual. This cell does not divide until it resumes meiosis and remains in a special chromatin status during the dictiate stage of the prophase from oocyte formation in the gonad in the female fetus until it dies through apoptosis or proceeds to ovulation. This status requires unique features to allow transcription to be active during oocyte growth and an automatic pilot system to drive the transition from tetraploidy to haploidy during maturation and fertilization and back to diploidy, all this in a few days in large mammals. Therefore, the regulatory program for all the transformations required for chromosome separation, cell cycle progression, response to sperm entry, and embryonic genome activation must be stored in the oocyte prior to ovarian release or even prior to final chromatin condensation as it inhibits further transcription. The data related to gene regulation during this period is limited for two main reasons: limited amount of material to study in mammals and differences with somatic tissues where gene pathways are much better characterized. Nevertheless, using the genomic amplification approaches and the increasing amount of information in somatic tissues and in oocytes from lower species, it is becoming possible to study this automatic pilot system that drives the mammalian oocyte through maturation-fertilization and embryonic genome activation. This chapter will focus on the progression of our understanding of the oocyte using proteomic and transcriptomic tools.
Meiotic Regulation by Maturation Promoting Factor and Cytostatic Factor in the Oocyte
Page: 80-92 (13)
Author: Gian Luigi Russo, Stefania Bilotto and Francesco Silvestre
In most of vertebrates, mature oocytes arrest at the metaphase of the II meiotic division, while some invertebrates arrest at metaphase-I, others at prophase-I. Fertilization induces completion of meiosis and entry into the first mitotic division. Several experimental models have been considered from both vertebrates and invertebrates in order to shed light on the peculiar aspects of meiotic division, such as the regulation of the cytostatic factor (CSF) and the maturation promoting factor (MPF) in metaphase I or II. Here, we reviewed the role of CSF and MPF and their biochemical pathways in regulating meiosis completion. Differences and similarities existing within several model systems of invertebrates, such as ascidians, cnidarians, mollusks, starfish, will be analyzed and compared to meiotic regulation in Xenopus. Data will be analyzed at the light of the phylogenetic conservation of MPF and CSF functions, accordingly to the position of these organisms in the evolutionary tree.
Gamete Binding and Fusion
Page: 93-103 (11)
Author: Young-Joo Yi and Peter Sutovsky
Fertilization is a stepwise process that starts before the actual gamete binding and fusion. Spermatozoa travel through the female reproductive system and respond to chemotaxis and thermotaxis to reach the oocyte. In most mammals, zona pellucida (ZP) glycoprotein ZPC is the primary sperm receptor that mediates sperm-ZP binding and acrosomal exocytosis (AE). During AE, the outer acrosomal membrane, already primed for AE during capacitation, fuses with the plasma membrane and undergoes vesiculation. The acrosomal matrix (AM) is exposed and dispersed in a step-wise manner. Sperm-ZP penetration is supported by sperm motility and by enzymatic activity of the hypothetical egg coat “lysin”, an acrosomal protease that digests the fertilization slit. Acrosin has been considered as a zona lysin candidate. However, male mice lacking the Acr gene are fertile. Recently, researchers have been focusing on the 26S proteasome as a mammalian and non-mammalian egg coat lysin. Following zona penetration, spermatozoa reach the perivitelline space and adhere to and fuse with the oolemma. Tetraspanin superfamily members CD9 and CD81 appear to act as sperm receptors on the oolemma, possibly supported by integrins and other elements within the cortical tetraspanin web. IZUMO, a member of immunoglobulin superfamily is a sperm ligand candidate for oolemma tetraspanins. Both CD9 and IZUMO are essential for gamete adhesion and fertility in the mouse. However, there is no evidence yet supporting the involvement of IZUMO and CD9 in sperm-egg plasma membrane fusion. After sperm-oolemma fusion, the fertilizing spermatozoon is incorporated into the ooplasm, a process aided by oocyte cortex microfilaments.
Ionic Events at Fertilization
Page: 104-120 (17)
Author: Brian Dale and Martin Wilding
The difference in ionic balance between the cytoplasm and the extracellular environment in all cells is maintained by ion channels and transporters located in the plasma membrane. In this chapter we review the voltage-gated channels and transporters common to both gametes and somatic cells and describe gamete specific ligand-gated channels in spermatozoa and oocytes. In lower deuterostome oocytes the fertilization potential is biphasic. The first event may be the result of gamete fusion, while the second larger depolarization is the result of the activation of several hundred non-specific channels in the oocyte plasma membrane. The fertilization channel is one of the largest channels known in biological membranes with a single channel conductance of upto 400pS and a reversal potential of +20mV. A soluble sperm factor appears to gate this channel via the ADPr/NO pathway. In higher deuterostomes the fertilization channel is a calcium-gated potassium channel. The type, number and topographical distribution of channels and transporters change continually during gameteogenesis, through fertilization to early embryonic cleavage stages indicating both the importance of ionic homeostasis and the role of second messengers in early development.
Recent Advances in the Understanding of the Molecular Effectors of Mammalian Egg Activation
Page: 121-134 (14)
Author: Christopher Malcuit and Rafael A. Fissore
Fertilization is the process by which male and female gametes, and their respective haploid genomes, fuse to form a single cell (the zygote) containing a complete diploid complement of genetic material representative of each parent. The male gamete, the sperm, transduces an activation stimulus to the awaiting female gamete, the egg (also, oocyte or ova), initiating a cascade of events collectively referred to as egg activation. The mechanisms of this process are both complex and tightly coordinated in order to impart a high level of fidelity necessary for initiation of embryonic development and propagation of the species. Although mechanistic discrepancies of this event exist even between species of the same phyla, calcium is the predominant driving force of egg activation in all species studied to date, and is responsible for promoting the resumption and exit of meiosis and the initiation of the developmental program. This chapter will focus on recent discoveries of the molecular mechanisms of egg activation with specific regard to the regulation of calcium signaling as it appears in the oscillatory mammalian system.
In Vitro Fertilisation
Page: 135-148 (14)
Author: Kay Elder
The historic birth of Louise Brown in 1978, the world’s first “test tube baby” justifiably ranks as a major milestone in the history of both medicine and science. This remarkable achievement represented the culmination of several different lines of research and investigation carried out by Patrick Steptoe, Robert Edwards and their respective colleagues: (i) More than two decades of laboratory research into the science of oocyte maturation and fertilization; (ii) Clinical observation and studies of the endocrinology and physiology of ovulation and implantation; (iii) Technological advances in the use of laparoscopy to observe the pelvic organs and rescue mature oocytes just prior to ovulation.
On the medical side, the results of their unique achievement offered treatment to couples who previously had no hope of having a child of their own; scientifically, the ability to initiate the creation of new life in the laboratory brought a revolution in biotechnology, and opened new vistas in our understanding of cell biology, the regulation of cell growth, and the events and control mechanisms surrounding fertilization and early preimplantation embryo development. This chapter will outline the current steps and procedures that are required for the successful establishment of a pregnancy when a couple undertake a cycle of assisted reproductive treatment by In Vitro Fertilization.
Current State of the Art in Large Animal Cloning: Any Lesson?
Page: 149-155 (7)
Author: Pasqualino Loi and Grazyna Ptak
Following fertilisation, major changes occur in the organisation of chromosomes and genes in the zygote that contributes to the formation of totipotent cells and their ensuing differentiation to the diverse cell lineages in the organism. The aim of animal cloning through Somatic Cell Nuclear Transfer (SCNT) is to reestablish the reprogramming that occurs in the fertilized embryo. SNCT often leads to abnormal development with a majority of concepti lost before birth. Imprinted genes are amongst the most frequently affected genes, and their altered expression could in part explain the observed developmental phenotypes. Here, we report our experience in sheep cloning. We describe the major phenotypic abnormalities observed in the extraembryonic tissues of sheep clones and suggest some possible strategies to improve SCNT.
Stem Cells from Oocytes and Oocytes from Stem Cells
Page: 156-166 (11)
Author: Fulvio Gandolfi, Georgia Pennarossa, Arianna Vanelli, Mahbubur M. Rahman and Tiziana A.L. Brevini
Full oocyte competence is the indispensable requisite for embryonic development and, at present, no ways are known to restore competence if, for any reason, it has been even slightly compromised. The same is not true for sperm cells that can initiate and sustain development even if severely abnormal or damaged as long as the DNA is intact. This points clearly to the uneven burden carried by the two gametes and indicates clearly the essential role of the oocyte. Parthenogenesis is the obvious consequence of such disparity with a number of lower species capable of giving birth to new individuals without any paternal contribution. Mammals are an exception to the rule due to epigenetic mechanisms limiting the parthenotes developmental potential. However, the blastocyst stage is easily reached and parthenogenetic stem cells can be generated whose differentiation potential seems to be much wider than that of whole parthenotes. Switching perspective, we move from stem cells originated from oocytes to oocytes originated from stem cells. Since embryonic stem cells can colonize the germ cell lines when chimeras are generated, it was not so surprising that oocytes can be obtained from stem cells. Although there is still a long way to go before full competence is reached it clearly opens the way to the hypothesis of having an unlimited source of oocytes. Finally, a recent and highly controversial set of results suggests that oocytes are not in such a limited supply as it is generally believed but post-natal oogenesis takes place at a surprisingly high rate.
Page: 167-175 (9)
Author: Elisabetta Tosti and Raffaele Boni
Events of reproduction occurring from meiotic resumption of the immature oocyte up to its exit from the second meiotic block following activation will be revealed. Morphological modifications of the oocyte during maturation will be related to signal transduction mechanisms involving primary or secondary messengers. A detailed view will be addressed to genetic and epigenetic control of oocyte maturation. At fertilization, reciprocal gamete activation and novel knowledge related to sperm factor and cascade mechanisms occurring in the oocyte will be focused. A detailed description of ion currents occurring during fertilization will depict another point of view of oocyte activation mechanisms, further supporting for their complexity. Finally, all basic information related to this short time lapse will be considered in relation to clinical application of assisted reproductive technologies. New frontiers, such as stem cells and cloning technologies, will be analyzed and future applications and improvements will be hypothesised.