Lecture 18: Gametogenesis and Embryological Development (Week 1)

Understanding Gametogenesis and the Cell Cycle

• Cell cycle understanding is key to understanding gametogenesis.
• The cell cycle has several checkpoints:
  • To ensure the integrity of replicated DNA
  • To verify chromosomes are correctly attached to spindles
  • To confirm that chromosomes are properly separated
  • To assess the quality of replicating DNA
  • To verify the integrity of DNA
• The cell cycle also includes a 'point of no return', a physiological state dependent on cell size

Cell Cycle Basics

• Preparation for DNA synthesis occurs in the G1 phase.
• DNA replication takes place during the S phase.
• The G2 phase involves further preparation for cell division
• Actual cell division (mitosis) occurs during the M phase.
• Cells can enter a state of quiescence or differentiation from G1, known as the G0 phase.
• Interphase comprises G1, S, and G2 phases

Chromosomes and Cell Division

• Somatic cells contain 23 homologous pairs of chromosomes, for a diploid number (2n) of 46.
• Autosomes consist of 22 matching pairs
• Sex chromosomes comprise one pair, either XX or XY.
• Gametes (oocytes and sperm) contain 23 chromosomes (1n).
• Mitosis is somatic cell division resulting in two genetically identical, diploid cells via single division.
• Meiosis is gem cell division, and results in four genetically different, haploid cells via two divisions

Mitosis vs. Meiosis I

• Mitosis involves DNA replication, followed by the separation of sister chromatids into daughter cells.
• Meiosis I begins similarly, but with synapsis and recombination between homologous chromosomes.
• In mitosis, there is no synapsis or recombination
• Meiosis I results in separation of homologous chromosomes.
• Metaphase I is when homologs align
• Anaphase I is when homologs separate

Meiosis II

• Meiosis II, daughter cells proceed to metaphase II, where chromosomes align
• Anaphase II is when chromatids separate, eventually leading to haploid daughter cells
• Mitosis = one division
• Meiosis = two divisions

Gametogenesis Initiation

• Gametogenesis starts with Primordial Germ Cells (PGCs) that produce gametes (sperm & oocyte).
• PGCs originate in the epiblast during the second week of embryonic development.
• PGCs migrate to developing gonads during the 4th-5th week.
• Mitosis increases PGC numbers.
• Meiosis generates both male and female gametes.

Oogenesis

• Once PGCs reach the female gonad, they differentiate into oogonia.
• Oogonia mitotically divide, reaching peak numbers in the ovary by the 5th embryonic month.
• Oogonia begin to stop dividing in the 3rd month and arrest in prophase of meiosis I, becoming primary oocytes.
• Some oogonia and primary oocytes undergo cell death, known as atresia.
• Surviving primary oocytes enter/arrest in prophase of meiosis I at the diplotene stage (resting), until puberty.
• Primary oocytes surrounded by flat follicular cells (epithelial origin) are called primordial follicles and are present at birth.
• Follicular cells secrete Oocyte maturation inhibitor (OMI) which maintains primary oocytes in diplotene stage.
• At puberty, 15-20 primary oocytes/follicles complete meiosis I each month (~28 days).
• Meiosis I completion generates 2 unequal cells: a secondary oocyte and a first polar body.
• The secondary oocyte starts meiosis II, but pauses in metaphase before ovulation.
• Meiosis II completes only if the oocyte is fertilized, which results in a fertilized oocyte and a second polar body
• Unfertilized oocytes die.

Oogenesis - Supporting Cells Details

• Primary oocytes have cytoplasmic contents (cortical granules) and are surrounded by flattened cells
• A primordial follicle: Primary oocytes wrapped in flat follicular cells of epithelial origin.
• Primary follicle: Primary oocytes (- cytoplasmic contents mature, free ribosomes, Golgi, etc.) wrapped in cuboidal-to-stratified cells.
• Secondary (antral) follicle: A fluid-filled cavity: the antrum is formed from granulosa cells that produce OMI which inhibit oocyte growth
• Mature (Graafian) follicle: Secondary oocyte + polar body. Granulosa cells are now termed Corona radiata formed from ovary interstitium.
  • This is a Large follicle that bulge out the ovarian cortex

Spermatogenesis

• Spermatogenesis is a developmental event that transforms spermatogonia into spermatozoa
• It begins at puberty, and occurs in the seminiferous tubules (testis)
• Three phases of spermatogenesis: Spermatogonia, Spermatocyte, Spermatid (Spermiogenesis).
• It takes 74 days for a spermatogonium to become spermatozoa.
• PGCs generate spermatogonial stem cells via mitosis.
• Production of type A Spermatogonia is the initiating event in spermatogenesis
• Type Ad (dark) Spermatogonia generate more type Ad and Ap (pale) Spermatogonia.
• Type Ap Spermatogonia generate more type Ap Spermatogonia and remain connected via cytoplasmic bridges.
• Type Ap spermatogonia differentiate into type B spermatogonia.
• Appearance of Type B Spermatogonia indicates this phase is over
• Type B Spermatogonia enter meiosis I and become primary spermocytes.
• Primary spermocytes pause (about 22 days) in prophase, then complete meiosis I to form secondary spermatocytes.
• Secondary spermatocytes generate spermatids via meiosis II.
• Cytokinesis remains incomplete (type A spermatogonia to spermatids); cells connected via cytoplasmic bridges, so all cells are generated from a type Ap spermatogonia cell, a clone that is kept in sync.

Spermatid Phase

• Spermatid (Spermiogenesis) phase is the process by which round spermatids are transformed into spermatozoa
• It has 4 phases
  • Golgi phase: acrosomal vesicle forms, axoneme initiated
  • Cap phase: acrosomal vesicle - acrosomal cap forms, nucleus condenses
  • Acrosome phase: manchette forms & disappears; neck, middle piece & tail form
  • Maturation phase: cytoplasm is extruded, Sertoli cells phagocytose cytoplasmic bridges releasing spermatids
• Acrosome is formed by the Golgi apparatus, contains soluble enzymes (hyaluronidase, hydrolases & proteases, acrosin), position determines anterior pole
• Flagellum is made of a central skeleton of 11 microtubules (axoneme). It is wrapped in a thin cell membrane, and an accumulation of mitochondria;
  • It is enables sperm to move at 1-4mm/min in fluid medium

Spermiogenesis Details

• In epididymis fluid: Spermatids rely on fluid (Sertoli cells) and peristaltic contractions to move to the it.
• In epididymis, maturation events include: acquiring motility, DNA condenses, sperm head size decreases, additional reduction of cytoplasm
• Mature also involves acquisition of a mature complement of plasma membrane proteins, and glycosylation
• Finally it becomes a surface-associated decapacitation factor added to sperm head which inhibits fertilisation abilty

Hormonal Control of Gametogenesis: Spermato & Oogenesis

• Testosterone (secreted by Leydig cells) promotes growth and division of Spermatogonia
• Leutinizing hormone, LH (secreted by anterior pituitary) stimulates Leydig cells to produce testosterone
• Follicle-stimulating hormone, FSH (secreted by anterior pituitary) stimulates Sertoli cells to promote sperm maturation.
• Growth hormone promotes early divisions of spermatogonia
• Estrogens are formed by Sertoli cells, and contribute to spermatogenesis.
• During oogenesis:
  • Follicle-stimulating hormone (FSH), secreted by the anterior pituitary, stimulates growth of follicles and promotes LH receptor expression on granulosa cells.
  • Leutinizing hormone (LH), also secreted anterior pituitary, stimulate thecal cell proliferation and secretion, stimulating ovulation.
  • Estrogen (secreted by granulosa cells) fills the antrum and upregulates its own FSH receptor and increases LH receptor expression and overall thecal cell proliferation.

Problems in Gametogenesis - Nondisjuction

• Nondisjunction: failed separation of chromosomes, primarily occurs in meiosis
• Abnormal chromosome number (occurs in mitosis or meiosis)
• Trisomy (chromosomal gain): Klinefelter syndrome (XXY) is an example
• Monosomy (chromosomal loss): Turner syndrome (45, X) is an example
• Translocations: Down's syndrome (chromosomes 14 & 21, extra 21)
• Common between Chromosomes 13, 14, 15, 21 & 22 (cluster during meiosis)
• Chromosomal deletions (large or micro)
  • Microdeletions: Angelman's syndrome (maternal deletion 15q), or Prader-Willi syndrome (paternal deletion 15q) - Large deletions: Cri-du-chat syndrome (chromosome 5p)

Aneuploidy Example: Kleinfelter vs Turner Sydrome

• Klinefelter (XXY): tall, long lower limbs. Also small testes, gynecomastia.
• 75% cases result from paternal non-disjunction during first division
• Turner (XO): short stature, webbed neck, absence of sexual maturation
• 75% cases result from paternal X chromosome missing

Aneuploidy Example: Trisomy 21 Incidence

• Cognitive deficiency; brachycephaly, flat nasal bridge; upward slant to palpebral fissures; protruding tongue, palmar flexion crease, congenital heart defects
• Likelihood increases with maternal age
• The incidence of trisomy 21 at fertilization is greater than at birth; 75% abort, and at least 20% stillborn

Quantity of Female Gametes

• 5th embryonic month has 7 million oogonia.
• Birth has 600,000-800,000 primary oocytes
• Puberty has 40,000 primary oocytes
• At the end of fertility period typically 500 primary oocytes are ever ovulated
• Each month 15-20 follicles start to mature

Quantity of Male Gametes

• Approximately 300 million mature sperm produced each day but with up to 10% detectable defects
• Defect types are in head, neck, mid-piece and tail.

Gametogenesis: Comparison

• Primary oocytes undergo DNA replication before meiosis in females, while primary spermatocytes also do the same in males.
• Both go through First, and Second Maturity Division which results in Polar bodies or Spermatids

Embryonic timeline

• Embryogenesis is the process of which cell starts to change into a baby.
• Embryogenesis spans from 1st 8 weeks of human development
• Fetal period spans from end of week 8 until birth
• Gestational age is considered pregnancy and spans 40 weeks of gestation from LNMP : last normal menstrual period
• Embryonic age spans 38 week gestation but begins at fertilization approximately 2 weeks after LNMP
• Week 1 is fertilization to about day 7

Embryological Development Week 1 Events

• Fertilization
• Blastomere formation
• Morula formation
• Inner cell mass and outer cell mass formation
• Blastocyst formation
• Attachment to endometrium

Female Gamete Preparation & Egg Transport

• Surge in Luteinizing hormone (LH) triggers oocyte to complete meiosis I and initialte meiosis 2
• The oocyte gets released (ovulation) along with attached granulosa cells
• Egg travels into the ampulla.
• Sperm must undergo capacitation for approximately 7 hours within the female reproductive tract.
• Female system assists sperm movement via: direct swimming, peristalic contractions and ciliary moment

Fertilisation & Prevention of Polyspermy

• In general sperm deposited in upper vagina/cervices only have a few sperm neary the oocyte
• Hyaluronidase, and proteolytic enzymes are released to penetrate granulosa cell layers forming cumulus layer.
• Upon reaching the zona pellucida, they adhere specifically with GalT and causes acrosome reaction
• Acrosomal enzymes digest zona pellucida to create a passageway
• When plasma membrane of sperm and oocyte adhere calcium ions enter the oocyte
• Intracellular Ca2+ triggers glyco-conjugates, proteases, glycosidase via exocytosis to form cortical reaction

Dosage Compensation

• In mammals, sperm and egg fusion yields either XX or XY sex chromosomes.
• Two X (XX) chromosomes result in too much X chromosome gene prodcuts, unless one is switched off (X dosage comepnsation)
• First the X chromosome condenses and becomes inactive within the cell
• This X chromsosome inactivation can result in a Calico pattern of alternating colours on the X chromsosomes

X Chromosome Inactivation Timeline

• The occurance is a two step event that requires XIC, Xist, and Tsix.
• The XIC (X-inactivation Center) is a specific part of the X chromosome with two genes of Xist and Tsix
• In all cells prior to implantation Tsix inhibits random X inactivation
• In all inner cells either paternally Xp, or matenallyl derived (Xm) get condensed becoming inactive
• There are two types with a non randomly Imprinted copy Inavtivation (iXCI) that happens first.
• There is then more Random X Chromosome Inactivation (rXCI) which then follows.