C2006/F2402 '06 -- Outline of Lecture #23. Power Point slides for this lecture are posted on Courseworks.

(c) 2006 by Alice Heicklen; Notes courtesy of Jeff Farrell. Last update 05/01/2006 01:52 PM

I. Cell Fate Determination

• Stages of specification of a cell:
  • Pluripotent – can become any cell in the embryo.
  • Committed  -- programmed to become one thing but could be reprogrammed
  • Determined – locked into a particular fate
  • Differentiated – now has obvious phenotypical difference
• Experiment to tell difference between commitment and determination:
  • Transplant some back tissue to the stomach of an embryo at its blastula stage
  • Does it become stomach or back?
    • If becomes stomach – Cells were only committed and can be reprogrammed
    • If becomes back – Cells were already determined and cannot change to becoming stomach
  • Determination does NOT mean that there was an obvious phenotypical difference necessarily – that will happen later when the cell differentiates.
• Gametes are a special cell type: they remain pluripotent, though they become determined – they can form any cell type (as they must, once they become an embryo), but yet are determined to become gametes.

II. Stem Cells & Cloning

• Adult stem cells – necessary to maintain some tissues of the body, for ex:
  • Intestines
  • Blood
  • Hair Cells  
  • Sperm (the average male produces 2 million sperm per day)
    

• Frog cloning
  • Briggs & King in 1950s
  • Easiest to do in frogs because they use external fertilization – don’t have to get an egg out and then reimplant it
  • The nucleus of a terminally differentiated cell of an albino frog was placed into an oocyte of a wild-type frog that had its nucleus removed with a pipette (Removing the nucleus is called enucleation and results in an enucleated oocyte.)
  • The enucleated oocyte + transplanted nucleus produced albino frogs, even though it was the oocyte of a wild-type (colored) frog.
  • This proved 2 things:
    • ALL DNA is still present in the terminally differentiated cell.  It must be somehow turned off because the cell is no longer pluripotent, but it is not lost.  (It has “genomic equivalence” with the gamete.)
      • (Can you think of an exception to this?  Would a differentiated B lymphocyte work?  Why or why not)
    • The differentiated nucleus can be reprogrammed to become pluripotent again
• Sheep cloning (Dolly!)
  • This time you have to extract and then implant an egg – tougher.
  • Could use any terminally differentiated cell as source of nucleus, but here used one from the udder
  • The nucleus of an udder cell from the white sheep was put into the enucleated oocyte of a black sheep (this is called “somatic nuclear transfer”), and then implanted into the black sheep
  • A white sheep was born, so it was expressing the same proteins as the original, white sheep
  • HOWEVER, Dolly’s mitochondria came from the black sheep – remember that mitochondria have their own DNA and produce some of their own proteins… these would have come from the oocyte and would NOT have been transferred with the nucleus.
    • This means Dolly is NOT a perfect clone in the sense that she is not a perfect (genetic) replica of the animal that donated the nucleus -- all her nuclear DNA is the same as the DNA of the donor, but her mito. DNA is different. 
  • This experiment (along with the frog cloning one) highlighted one of the unique properties of the oocyte: its ability to reprogram DNA.
     
• Cloning Complications
  • X-chromosome Inactivation
    • Barr bodies are chosen at random, thus the same X-chromosomes will not necessarily be inactivated in the same pattern in the clone, which can have consequences.  (Such as different coloration in pigmented areas of the female cat.)
  • Imprinting
    • 30+ imprinted genes are known in humans
    • An imprinted gene is one that is differentially expressed depending on whether it came from the father or mother
      • This is NOT related to sex-linked traits.
    • For imprinted genes, methylation is added to the DNA in the process of gamete formation. The sites of addition during spermatogenesis are different than the sites of methylation during oogenesis. The methylation pattern allows the cell to know which copy is maternal and which is paternal.
      • Somatic nuclear transfer will result in only the oocyte patterns being put on when the oocyte reprograms the nucleus
  • Telomeres
    • Repeating DNA at the ends of linear chromosomes
    • Some is lost during each replication
    • Act as a bit of a “biological clock” – cells cannot divide if telomeres become too short
    • Telomerase, a protein, is expressed during gametogenesis and it restores the telomeres
       
• Cells have “genomic equivalence”—their DNA is the same
  • However, only a small percentage of the genome is expressed; “housekeeping” genes are expressed in all cells; cells also express proteins specific to their cell type making them different from other cell types
  • This is because of transcriptional regulation that prevents the whole genome from being used
  • Much of this regulation is done via chromatin structure (remember heterochromatin and euchromatin?) Tighter chromatin is transcribed less.
    • These chromatin structures are remembered through mitosis.
    • Need special environment of the oocyte to remove these modifications
       
• Obtaining human stem cells (Handout 23A)
  • ES cells = embryonic stem cells
  • Can grab inner cell mass of the blastocyst
  • Can grab the fetus’ primordial germ cells (PGCs) which will become gametes, but are pluirpotent
  • Adult stem cells - advantage: do not need to distroy embryo
  • Somatic nuclear transfer
    • “therapeutic cloning”
    • Avoids the problems of immune rejection

III. Life Cycle

• The Frog as a representative life cycle (Handout 23B)
  • The organism must be able to function throughout development
    • Need a heart quickly, as the embryo increases in size and diffusion is no longer sufficient to bring O₂ in and CO₂ out.
  • Sperm seen as DNA made motile by a flagellum
  • Egg has lots of cytoplasm (or yolk) filled with nutrients (unlike sperm)
  • Fertilization spurs a wave of calcium ion from the ER into the egg cytoplasm
  • Cleavage begins
    • For a while, have very rapid cleavages without increasing size of the embryo
      • (Skipping G1 and G2 phases of the cell cycle, and going directly from S to M back to S…)
  • Morula = 16-64 cell stage (in frog) = stage when embryo is still solid, before cavity forms.
  • Blastula = After fluid filled cavity (blastocoel) forms in the middle
    • Allows cells to begin to move relative to each other (gastrulate)
    • Separates cells at top of blastocoel from bottom of blastocoel
      • Prevents paracrine signaling between those cells
  • Cells begin to move (gastrulate) directly opposite point of sperm entry
  • THREE GERM LAYERS:
    • Ectoderm – covers the embryo, and will form the epidermis, CNS, and so on.
    • Endoderm – inside of the embryo – forms the gut and respiratory systems
    • Mesoderm – between the endoderm and ectoderm – forms most of the major internal organs, including the heart, muscle, bones, kidneys, and blood
  • Organs begin to form
    • First the heart, to allow for circulation
    • Next the CNS
  • Eventual hatching when outer membrane (the chorion) is lost

IV. Gametogenesis

• Sex chromosomes determine the identity of the gonads.  These gonads then shape the primodial germ cells into becoming the proper gametes for that sex.
  • Males form Sertoli cells (nurture sperm during spermatogenesis) and Leydig cells (which produce testosterone)
  • Females form thecal cells and granulosa cells
  • SRY gene on Y-chromosome responsible for these differences – its expression causes the bipotential gonad to form male structures (Leydig and Sertoli cells).
     
• Spermatogenesis
  • Sertoli cells form the tubules of the testes
    • Cadherins attach sperm to Sertoli cells
    • Sertoli cells secrete a basal lamina
  • In between the Sertoli cells are Leydig cells
  • Sperm move farther and farther from the basal lamina as they mature
    • Takes 65 days for a sperm to mature in humans
  • A1 (stem) cells divide into 2 – one cell to replenish the A1 population and one to become sperm
  • The dividing cell goes through mitosis repeatedly, forming a multinucleated cell (it never fully divides its cytoplasm)
  • This becomes a type B cell, which will undergo 1 mitotic division, then meiosis
    • Primary spermatocyte goes through Meiosis I, to give secondary; Secondary spermatocyte goes through Meiosis II to give spermatids, connected by cytoplasmic bridges. See Purves, fig. 43.3, or Becker fig. 20-10, or Sherwood Fig. 20-7 or Kimball
  • Eventually the cells differentiate to look like sperm
    • They get rid of as much cytoplasm as possible, which is left as “residual bodies.”
    • The centriole organizes the sperm’s flagellum (which is made of MTs)
      • Dynein motors move the flagellum by sliding the MTs against each other.  Since they are anchored down at the bottom, this results in bending of the flagellum. (Handout 23A middle)
      • Animation of sperm's flagellum structure and movement by dynein motors
    • The acrosomal vesicle forms, which contains proteases needed to penetrate the egg during fertilization
    • Mitochondria line up around the flagellum
    • DNA is crystallized through removal of histones and their replacement with proteamines
    • XY bodies prevent transcription and translation during spermatogenesis

See next lecture for oogenesis.