Student Reader

Sexual reproduction

Requires joining of two haploid cells (gametes) to form a diploid individual. Gametes are produced by gametogenesis, a process involving meiotic cell divisions. Two meiotic events contribute to genetic diversity: crossing over between homologous xsms and independent assortment of xsms. Sexual reproduction itself contributes much genetic diversity, because the two parents have tremendous xsmal disparity. There are three fundamental steps:

  • Gametogenesis (making gametes)
  • Mating (getting together gametes)
  • Fertilization (making gametes fuse)

Gray crescent is where sperm-binding sits are located. When the sperm enter the cytoplasm rotates toward site of sperm entry. This reveals band of pigmented cytoplasm on egg opposite sperm entry site. This is called the grey crescent.

Rearrangements of egg cytoplam set stage for determination. β-catenin is a crucial transcription factor from materanl mRNA found through cytoplasm. Also protein kinase GSK-3, which phospohorylates and inactivates β-catenin. In the vegal cortex of egg is GSK-3 inhibitor. This inhibitor is oved along microtubules to the gray crescent where it keeps β-catenin from being ddegraded.

Cleavage: rapid cell divisions leading to bundle of cells is called cleavage. IThe cytoplasm is not homogeneous so distriubition of nutreints plays a role in determinants of cells of early embryo. No cell growth and little expression just replication. This balls forms a central fluid-filled cavity blastocoel (morula is just the ball of cells). The embryo is now a blastulate with individual cells called blastomeres.

The amount of yolk influences cleavage. IN embryos with little or n yolk then cleavage furrow formation is simple. More yolk means more resistance to cleavage furro formation. The blastodisc results from incomplete furrow formation. Otherwise you have complete cleavage.

Orientation of mitotic spindles determines planes of cleavage and therefore arrangement of daughter cells.

Gastrulation: producing the body plan

  • Fertilization
    • The sea urchin is one of the several model organisms that has been used to decipher the basic cellular and molecular biology of animal fertilization.
    • More is known about fertilization in sea urchins than is known about fertilization in most vertebrates.
    • In animals in general, fertilization is the direct interaction and fusion of two germinal cells (one "egg" and one spermatozoan), resulting in the initiation of cleavage, gastrulation and the species-specific developmental program that characterizes each organism.
    • In sea urchins, a discrete series of steps characterizes fertilization (Vacquier, 1998).
  • Attraction of spermatozoa by fully mature ova
    • Peptides dissolve from the jelly coat and stimulate sperm respiration, motility and chemoattraction in a species-specific manner.
    • Sperm follow a decapeptide gradient in sea water towards the region of higher concentration at jelly coat of recently spawned fully mature ova.
    • These peptides have been recovered from egg-conditioned medium.
    • In S. purpuratus and Hemicentrotus pulcherrimus, the peptide is entitled speract (Gly-Phe-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly) and in Arbacia punctulata the peptide is called resact (Cys-Val-Thr-Gly-Ala-Pro-Gly-Cys-Val-gly-Gly-Gly-Arg-Leu).
    • Species specificity depends on the COOH end of the peptide and deletion of the Val or Val-Gly residues results in loss of respiration-stimulating activity.
    • A 767 amino acid protein that serves as a voltage sensitive channel in the plasma membrane of the spermatozoan has been isolated from the testis of S. purpuratus.
    • Such a channel might become activated by speract-induced hyperpolarization and lead to flagellar beating.
  • Activation & Acrosomal Reaction of Spermatozoa
    • Activation of the many spermatozoa which triggers the acrosomal reaction results from direct their contact with a highly sulfated fucose sulfate polymer in the jelly coat of the fully mature ovum.
    • This interaction causes adherence of many spermatozoa to the jelly coat and opening of incurrent Ca2+ channels in their cell membranes that are similar to human polycistin-L.
    • The acrosomal reaction results in fusion of the acrosomal vesicle membrane with the spermatozoan plasma membrane and permits the release or secretion of the contents of the acrosomal vesicle.
    • Hydrolytic enzymes from the acrosomal vesicle begin to digest constituents of the jelly coat.
    • Additionally, excurrent proton pumps in sperm membrane are also opened at this time and their activity leads to alkalinization of the cytoplasm in the head of the spermatozoan.
    • Alkaline cytoplasm promotes the loss of profilactin blocking protein from ends of actin monomers.
    • Explosive polymerization of actin generates microfilaments that extend a membrane covered acrosomal process from the bottom of the vacant acrosomal vesicle.
    • The acrosomal process projects through the remains of the jelly coat towards the vitelline membrane of the egg.
    • As this occurs, bindin (not a glycoprotein or a transmembrane protein) becomes associated in an unknown manner with the tip of the acrosomal process.
    • Sea urchin bindins are not related to any other proteins, but they do contain a central domain of 60 amino acids that has been conserved for over 150 MY.
    • Also released from the acrosomal vesicle at this time are syntaxin and VAMP which become involved in secretion of the contents of the granule granules of the fully mature ovum following fertilization.
  • Binding of the membrane of spermatozoa with the vitelline membranes of fully mature ova:
      Binding receptors on the vitelline membrane recognize and attach to bindin protein on the tip of the acrosomal process.
    • Sea urchin egg receptor for bindin is a complex molecule that is still very controversial.
    • The putative receptor is a single protein that has several domains and is unlike any known class of receptors.
    • On its carboxyl terminus it has a short cytoplasmic domain, followed by a single membrane spanning domain.
    • Extracellular domains on the amino termins include a potential vitelline membrane spanning domain followed by a domain that is similar to cytoplasmic heat shock 70 proteins in the vertebrates.
    • Since the function of heat shock proteins is to bind to and fold other proteins, perhaps this HS70 domain can bind bindin.
    • Subsequent to these analyses, other researchers have disputed the validity of this interpretation, even suggesting that the protein identified as the bindin receptor is actually a soluble, cytoplasmic HS110 protein.
    • Resolution of this issue will await further sequence data.
  • Fusion of the cell membrane of the spermatozoan with that of the fully mature ovum:
    • While thousands of spermatozoa can potentially achieve this level of interaction with the fully mature ovum, the vitelline membrane is finally breached by the acrosomal process of a single spermatozoan.
    • The membrane of this spermatozoan fuses with that of the fully mature ovum.
    • Why only one spermatozoan is capable of this act, when many are marsheled to accomplish the same task is one of the unanswered questions of sea urchin biology.
  • Activation of the cytoplasm, the cortical reaction of the fully mature ovum and assembly of the fertilization envelope:
      After fusion, the metabolism of the egg changes radically during activation.
    • The membrane of the fully mature ovum is depolarized radially away from point of fusion.
    • Gamete membrane fusion elicits a wave of Ca2+ release from intercellular stores that initiates at the point of fusion and causes exocytosis of some 15,000 cortical granules.
    • Included in these granules is
      • 1) hyalin which forms a layer immediately surrounding the egg,
      • 2) a colloid that raises the fertilization membrane by imbibing H20,
      • 3) a serine protease (CGSP1) that destroys receptors for sperm on the vitelline membrane,
      • 4) a protein termed vitelline delaminase that may cleave the connection between the vitelline membrane and the oolemma,
      • 5) a structural protein that in the presence of H202 is polymerized onto the inner surface of the old vitelline membrane, now called the fertilization membrane
      • 5) ovoperoxidase, a heme-dependent peroxidase that functions to block polyspermy by interacting with the structural protein mentioned above in the presence of H2O2 to form the new fertilization membrane
    • The increase in respiration within the cytoplasm of the newly fertilized egg produces H202 which serves as the oxidant for the catalysis of the structural proteins to the inside of the old vitelline membrane by dityrosine crosslinkages.
    • The consideralbe oxidative damage that could result from the presence of H202 evolved in this process is countered by an intracellular amino acid called ovothiol.
    • Ovothiol is oxidized to the disulfide thus consuming H202.
    • There results a highly protective shield, the fertilization membrane, that protects the fertilized fully mature ovum or zygote.
  • Pronuclear Fusion
    • Within the cytoplasm of the newly formed zygote, the centriole near the female pronucleus generates an array of microtubules.
    • Internalization of components of the sperm is accomplished using a myosin molecular motor and the microtubules of the acrosomal process.
    • The haploid male pronucleus and the centriole and some cytoplasm from the spermatozoan are drawn into the cytoplasm of the egg.
    • The nucleus of the spermatozoan swells and becomes the male pronucleus and its centriole generates an array of microtubules.
    • Female and male pronuclei fuse following their migration towards each along their respective microtubular arrays.
    • Either the female pronucleus migrates to the male or the male pronucleus migrates to the female and resulting fusion of the membranes of these pronuclei produces the diploid zygote nucleus.
    • The chromosomes from these nuclei have already undergone pre-meiotic S-phase during which they replicated their DNA.
    • Mitotic cleavage divisions follows and generate the blastula.
  • Cleavage
    • Cleavage is a mechanism for cloning the zygote genome generated at fertilization following male and female pronuclear fusion.
    • Usually in edible sea urchins, cleavage is oligolecithal, indicating that a limited amount of yolk (= nutritional molecules) is present in the cytoplasm mostly of the vegetal pole of the egg.
    • Many informational molecules, like the protooncogene c-myc are present in the cytoplasm in stored form, as untranslated mRNA with short polyA tails.
    • Intracellular signalling pathways that result in the first cleavage division are just now coming into focus and they bear a striking similarity to the initiation of the cell cycle in somatic cells.
    • There is a mechanism for the release of calcium. Account for a Ca2+ increase and alkalinization of the cytoplasm. Phospholipase Cgamma is involved in fertilization and cleavage. Calcium transient requires production of inositol triphosphate.
    • Yet not clear how MAP kinase pathway is involved. For example, c-myc is already present and this common denominator can upregulate expression of G1 cyclins that promote the cleavage cell cycle.
    • Maybe donÕt need the cytoplasmic phosphorylation cascade that accompanies induction of mitosis in somatic cells, since myc protein is already there. Otherwise this is one of the major functions of the MAP-kinase based phosphorylation cascade.
  • Gastrulation
    • Gastrulation is when many organisms begin to express the new embryonic genome formed during fertilization.
    • Up until this point, maternal informational molecules were used laid down during oogenesis.
    • Many urchins die at gastrulation as lethal combinations of genes produce either non-functional or defective proteins.
    • MYP is digested by cathepsin B in such early embryos. MYP was produced during the pregametogenesis and NP renewal stage and was transferred intact to primary oocytes during oogenesis.
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