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What I'd like to do today,
is to give you an epilogue,

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the very end of the last lecture on
cell type, that I did not want to

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cram into the very last one minute,
so I'm going to take five minutes

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now to talk about that,
and then I want to move on to a

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topic that is very related,
because everything, of course,

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is very much related by the control
of gene expression,

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by cell-cell interactions,
by cell fate, and so on, but I want

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to talk to you about a particularly
important set of interactions

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between cells.
Here we are, on your game board of

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life, we're moving up through the
formation module,

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talked about cell type,
and we're going to build on that

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conversation in talking about
fertilization today.

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So let me go back and remind you
where we were.

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At the very end of last lecture we
were talking about cell type,

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and what I wanted to do with you,
was to trace for you the ontogeny,

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the development of a particular type
of cell, and indicate to you how

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many different interactions there
were between cells --

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-- how many different sets of genes
had to be activated,

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and had to interact with one another,
to get you from a fertilized egg,

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all the way to a differentiated cell
type, and I used the paradigm of

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skeletal muscle.
And really, the point that I think

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you got, was that this is very
complicated, and if you go on in

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your careers and think about,
as engineers for example, as

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biologists, engineering a particular
kind of cell from another kind of

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cell, you really have to know these
steps along the way,

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and ask whether or not in order to
get from point a to point b,

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you have to go through the whole set
of 30 steps --

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-- or whether you can circumvent
those steps, and short-circuit,

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and go on a much shorter route.  And
we'll talk more about this in

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subsequent lectures,
but this is very relevant for

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thinking about how to engineer
particular cell types.

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What we were talking about was an
extraordinary experiment that was

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done in the 1920's,
that demonstrated a piece of

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embryonic tissue called the
organizer of the dorsal mesoderm,

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was able to activate the formation
of a second --

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[PAUSE[
-- axis, or a second conjoined

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embryo, where most of the second
embryo was derived from the host

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tissue, into which this piece of
organizer, or dorsal mesoderm,

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had been transplanted.  And this
organizer was able to induce a

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second embryo,
including skeletal muscle,

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must be made from the host cells.

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OK, who does not have their handouts
here from the last lecture?

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I'm not trying to nail you,
I just want to know if I need to

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write something on the board.
I need to write something on the

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board, OK.  So,
the notion that we were left with

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was that, until now,
we had gotten to the point where

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genetic information that had defined
the dorsal part of the embryo,

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synergized with genetic information
that formed the dorsal mesoderm that

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formed the mesoderm.
That gave rise to this piece of

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tissue called dorsal mesoderm,
abbreviated meso, which is also

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known as the organizer.
And this piece of dorsal mesoderm,

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or organizer, itself, as I said, a
very powerful inducing center,

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and it induces formation of a group
of tissue blocks called somites.

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These somites,
as I'll show you in a moment,

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are segments, or segmental units,
that are present along the whole

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axis of the body,
the whole trunk region of the body

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of vertebras.  And they actually
hark back to our ancestry,

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derived from organisms that were
fully segmented,

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worms for example,
earthworm type animals,

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that had multiple segments.
OK.  The somites,

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in turn, become subdivided,
and I put this in just to indicate,

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again, the complexity of the process,
and part of the somite,

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termed the myotome, activates the
expression of the transcription

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factor myoD.
And it was pointed out to me by

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Christine that I was abbreviating
transcription factor,

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TXN that is my abbreviation,
many of you may remember from way

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back, for transcription.
So, if you have in your previous

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notes, a TXN, and you're not sure
what I meant, I meant transcription.

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OK, so these somites get subdivided,
they make the myotome that expresses

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myoD, and as we talked about last
time, we come in full circle to

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where we wanted to be,
which is the activation of the

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skeletal muscle program.
Let me show you this on this

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PowerPoint slide.
OK, so here are the organizers in

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diagram, activating the formation,
or inducing the formation, of these

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somites.  The somites get divided,
myoD is expressed, myoD and other

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family members,
remember I made the point this was a

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member of a redundant transcription
factor, a redundant gene family,

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myoD maintains its own expression
through a positive alter

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regulatory route.
That means it binds to its own

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promoter, and perpetuates,
or activates, its own transcription,

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so once myoD is expressed, it
maintains its own expression,

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a very popular way of stabilizing
gene expression in particular cell

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types.  OK, here are some pictures
of early human embryos.

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Now, you should realize,
the reason in this previous slide

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that I went from a round circle
representing a round ball of cells

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as an embryo, to no circle,
is that everything I've told you

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until now, took place in a round
ball of cells,

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OK?  Up to about the 4,
00th cell stage, or about the 10,

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00th cell stage, the frog embryo is
just a round ball of cells,

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and most embryos are not very
distinguished looking.

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But the process of making somites
and dividing them,

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goes on until the embryo is a
million cells or more,

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and during that time, the embryo
reorganizes its body plan,

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so it's not longer a ball of cells,
it starts to take on its

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characteristic form.
And some of the characteristic form

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it takes on, is shown in this human
embryo, about 19 days after

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fertilization,
this region here is going to be the

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nervous system --
-- and on either side of the nervous

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system, I've got in red here,
boxed these little segmented blocks,

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and you can see these segmented
blocks in older embryos.

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Those are the somites from which
most of the skeletal muscle is going

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to form.  This is a movie to show
you the segmentation

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of the somites.
So here they go,

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here are the blocks of cells being
divided one from another,

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it's a very beautiful process,
and you can see it's very ordered.

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Down the middle here is the neural
tube, which is starting to give rise

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to the central nervous system,
and on either side of the neural

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tube is this future muscle tissue,
or the future somites that include

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the skeletal muscle,
and you can see them being divided

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symmetrically on either side of this
midline here, in to these

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chunks of tissue.
And it's actually a very interesting

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story in the regulation of gene
expression, as to how you time

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things in such a coordinated fashion.
In all animals,

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the somites become segmented first
in the head region,

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and then the segmentation moves down
towards the tail,

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and there is an intrinsic timer in
the embryo, that I don't have time

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to describe to you,
that again, converges on the

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regulation of gene expression,
that allows this extraordinarily

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coordinated segmentation
of the body plan.

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OK, so what I've done is to try and
trace this ontology of the cell type.

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What you'll realize if you think
about this, is that I haven't

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actually told you what transcription
factors actually activate the myoD

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promoter in the somites.
This is an RNA expression pattern,

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and the whole myoD regulatory loop,
is controlled in a transcriptional

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way.  And we don't know what exactly
activates myoD expression in the

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somites, but we know a lot about
what happens before.

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And with that,
I am going to move on to what I want

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to spend most of today talking to
you about, and that is the question

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of reproduction.
And I'm going to talk to you about

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the molecular biology of
reproduction, and particularly we're

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going to talk about sexual
reproduction --

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-- as one of the most important

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examples of cell-cell interactions.
And in fact, the one without which

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none of us would be here,
although that, in a sense,

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is a silly thing to say because
without most of the interactions,

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the cell interactions during
development, none of us would

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be here anyway.
But, this is a very interesting

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process, a very important process,
and it exemplifies some points that

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I want to bring up here.
So the notion here is that two

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haploid cells,
the sperm, and N is my designation

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for haploid, I presume you're all
with me when I use N and 2N,

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as haploid and diploid, OK, fine.
Sperm and egg go through the

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process of fertilization --

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-- to form a single cell,
a zygote that is diploid, and the

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zygote will then go on to perform a
multi-cellular embryo,

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etc.  Now, there are some really
extraordinary things about this.

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One of the extraordinary things is
that the sperm and the egg are fully

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differentiated cells.
I have been telling you,

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indeed really trying to hammer into
you, the notion that cells progress

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from less specialized states to more
specialized states.

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And this is the one instance during
development, where the flip is true,

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where the opposite is true.  So the
sperm and the egg are fully

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differentiated cells,
they have reached this final cell

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type, but when they fuse,
and they form the zygote, they now

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make the most fully undifferentiated
cell. So the zygote is uncommitted,

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that may or may not be spelled
correctly.

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So the zygote is uncommitted,
and I want to introduce you to a new

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term, which is that of potency,
where potency refers to the number

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of possible fates that a cell can
assume.  We're going to talk a lot

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about this when we talk about stem
cells.  The number of possible fates

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that a cell can assume,
and the sperm and the egg are

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uni-potent, they have one possible
fate, which is themselves.

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And the opposite is true of the
zygote.  It is totipotent,

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it can become everything, all
possible cell types.

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And that is the antithesis of what

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happens during development in
essentially, I would say,

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in every single cell type of the
body.  And it's extraordinary,

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and we don't understand, we don't
understand, how you get this

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complete reversal of fates,
although I'll try to go through this

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with you in a future lecture.
All right, why go to all this

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trouble?  This is a lot of work for
an organism to go through,

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and the bottom line is that it leads
to genetic re-assortment,

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and it leads to genetic diversity.
So the notion of sexual reproduction,

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I'm going to write it up here,
is that it engenders genetic

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diversity, and it does so because
genetic materials becomes

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re-assorted during meiosis,
and during recombination, or during

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the joining together, of the
sperm and the egg.

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OK, I divided this lecture up into a
few parts.  The first is

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gametal genesis --

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-- where the gametes are the egg and
the sperm, just a different name for

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them, and gametogenesis is one of
those developmental biology terms

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that has the suffix "-genesis",
the creation of the gametes.  Both

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the sperm and the egg start off from
diploid, precursor cells,

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and this is a very interesting story,
but I'm not going to tell you.

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But I'll remind you, well, I'm
going to allude to it.

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I'll remind you that long ago,
I showed you these beautiful series

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of pictures from worms.
When we talked about determinants,

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cell autonomous factors that control
cell fate, and I showed you how

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these determinants were segregated
into a single cell,

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of the 32 cell worm embryo,
and that this cell was going to give

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rise to the gametes, the
egg and the sperm.

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In fact, the exact same thing is
probably true in all animals,

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including ourselves.  And certainly
in frogs, and in fish,

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we know that there are determinants
that are segregated,

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and we know what these are,
they turn out to be proteins that

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bind RNA and control translation of
presumably specific target genes.

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So, somewhere back in the dawn of
time, during development,

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the germ cells become determined --
-- but I am not going to talk about

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this, I am going to talk about them
when they actually already know that

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they're germ cells,
and when they start thinking about

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assuming their final differentiated
fate.  And I want to make a couple

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of contrasting points,
and OK, well let me do my board work

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and then we can look at that.  OK.
The precursor of the spermatozoa is

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the spermatagonium.
This is a diploid cell that

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undergoes meiosis,
to give rise to a haploid

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spermatazoan.  The spermatagonium is
a moderate sized cell,

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it's just a regular sized cell,
and when it undergoes its conversion,

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its differentiation,
to a  spermatazoan, or a sperm,

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it becomes very small, it jettisons
most of its cytoplasm, and

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it becomes motile.
So you go from a medium sized cell

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to a tiny cell that has the very
important property of being motile.

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And this is true, really,
universally, some,

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and I'll talk about this in a moment.
The way that spermatozoa are motile

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is slightly different in different
organisms, but they pretty much,

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all spermatozoa are motile.
In contrast, the egg arises from a

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diploid oogonium and the oogonium
also starts off as a medium sized

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cell, but it does the opposite.
It gets very big, OK?  And the

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oogonium moves to change into
something called an oocyte,

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00:20:06,000 --> 00:20:14,000
or I prefer the term, egg, and this
was a haploid cell.  OK.

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00:20:14,000 --> 00:20:18,000
It's large, by necessity sessile,
and it's large because it stores a

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00:20:18,000 --> 00:20:23,000
bunch of stuff,
and the stuff that it stores is food,

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00:20:23,000 --> 00:20:28,000
to nourish the early embryo before
the embryo can fend for itself,

223
00:20:28,000 --> 00:20:33,000
or before it can be nourished by the
mother.  And it also contains

224
00:20:33,000 --> 00:20:38,000
components that regulate
early development.

225
00:20:38,000 --> 00:20:42,000
So it's full of things like
determinants.  It's also full of

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00:20:42,000 --> 00:20:46,000
machinery that can make ribosomes
and so on, and the size of the egg

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00:20:46,000 --> 00:20:50,000
varies from organism to organism.
In frogs, for example, the egg is

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00:20:50,000 --> 00:20:55,000
about a millimeter in diameter,
in humans the egg is about 50

229
00:20:55,000 --> 00:20:59,000
micrometers in diameter,
because the mother nourishes the egg

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00:20:59,000 --> 00:21:04,000
sooner than, or the embryo,
sooner in humans than in frogs.

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00:21:04,000 --> 00:21:08,000
All right, so let's look at a couple
of these diagrams that I gave you.

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This is to remind you about meiosis
and what meiosis is,

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00:21:13,000 --> 00:21:18,000
and I want to make a couple of
points that are up here on the top,

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00:21:18,000 --> 00:21:23,000
some of which are more important
than others.  Spermatozoa,

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00:21:23,000 --> 00:21:28,000
and spermatogenesis, is featured by
continuing mitosis throughout life

236
00:21:28,000 --> 00:21:33,000
of the male.  All the meiotic
products become sperm,

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00:21:33,000 --> 00:21:38,000
but this continuing mitosis is
what's really important.

238
00:21:38,000 --> 00:21:42,000
The spermatogonium,
the precursor of the spermatozoa,

239
00:21:42,000 --> 00:21:47,000
persists throughout life, and so you
get a lot of sperm produced.

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00:21:47,000 --> 00:21:51,000
So we can add to our list here,
that you get many sperm.  In humans

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00:21:51,000 --> 00:21:56,000
there are about five times ten to
the seventh sperm per ejaculate.

242
00:21:56,000 --> 00:22:00,000
If you go about five-fold lower
than that, you're in the range of

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00:22:00,000 --> 00:22:05,000
infertility.  We can talk about why
that is, and so one can do

244
00:22:05,000 --> 00:22:09,000
calculations, and it's been
calculated that males can make up to

245
00:22:09,000 --> 00:22:14,000
ten to the 13th sperm
in a lifetime.

246
00:22:14,000 --> 00:22:18,000
If you do the calculation,
you will understand that not all of

247
00:22:18,000 --> 00:22:23,000
those sperm are used,
and some are them are resorbed,

248
00:22:23,000 --> 00:22:28,000
so you can do that rather
interesting calculation another time.

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00:22:28,000 --> 00:22:33,000
OK.  Oogenesis,
in contrast to the plentiful and

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00:22:33,000 --> 00:22:38,000
cheap sperm, and we'll come back to
this point in a moment.

251
00:22:38,000 --> 00:22:43,000
In females, mitosis of the oogonia,
ends before birth, and in fact, by

252
00:22:43,000 --> 00:22:48,000
the time a child is born,
it is a female, it has about a

253
00:22:48,000 --> 00:22:53,000
million or so,
primary oocytes,

254
00:22:53,000 --> 00:22:59,000
which are still diploid,
they're along this pathway towards

255
00:22:59,000 --> 00:23:04,000
formation of the egg.
And by the time the child is about

256
00:23:04,000 --> 00:23:10,000
five, there are only about 500,
00 primary oocytes sites.  OK?

257
00:23:10,000 --> 00:23:14,000
And those cells have withdrawn from
the cell cycle,

258
00:23:14,000 --> 00:23:18,000
they are not making anymore of them.
So that is your pool from which to

259
00:23:18,000 --> 00:23:22,000
make eggs, and in fact,
most of those, 90% of those,

260
00:23:22,000 --> 00:23:26,000
are going to die, and you're going
to get a greater than 90% are going

261
00:23:26,000 --> 00:23:31,000
to die, and only about 500 eggs
mature during the lifetime of a

262
00:23:31,000 --> 00:23:35,000
human female.  OK?
And it's actually not quite clear

263
00:23:35,000 --> 00:23:39,000
why, but it's certainly harder to
make a good quality egg,

264
00:23:39,000 --> 00:23:43,000
probably, than to make lots of
smaller sperm,

265
00:23:43,000 --> 00:23:48,000
for reasons of energy expenditure of
the animal.

266
00:23:48,000 --> 00:23:53,000
All right, let's look through these
slides a little more.

267
00:23:53,000 --> 00:23:58,000
The testes is an extraordinary organ
that comprises many,

268
00:23:58,000 --> 00:24:03,000
many, many tubules, all coiled
together.  Within the seminiferous

269
00:24:03,000 --> 00:24:08,000
tubules there is an array of sperm
production that is architecturally

270
00:24:08,000 --> 00:24:14,000
very interesting.
Ignore this red up here for the

271
00:24:14,000 --> 00:24:19,000
moment, and look at this
architecture of the

272
00:24:19,000 --> 00:24:24,000
wall of one of the
The developing spermatozoa are

273
00:24:24,000 --> 00:24:29,000
embedded in cells called Sertoli
cells.  Sertoli cells supports,

274
00:24:29,000 --> 00:24:34,000
and nourish, and induce the
formation of the mature spermatozoa.

275
00:24:34,000 --> 00:24:40,000
So the spermatozoa that are most
immature, are lying away from the

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00:24:40,000 --> 00:24:45,000
luminal, the cavity,
of the seminiferous tubule,

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00:24:45,000 --> 00:24:50,000
and as these cells mature and
undergo meiosis,

278
00:24:50,000 --> 00:24:55,000
they move closer and closer to the
lumen, until you get a bunch of

279
00:24:55,000 --> 00:25:00,000
spermatozoa, with these tails
pointing out into the lumen of the

280
00:25:00,000 --> 00:25:06,000
tube, ready to be shed
upon ejaculation.

281
00:25:06,000 --> 00:25:10,000
So it's a very beautifully,
architecturally very beautiful

282
00:25:10,000 --> 00:25:14,000
process.  Now what does the
spermatazoan actually look like?

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00:25:14,000 --> 00:25:18,000
It's got three parts.  It's got
this head that's got a nucleus that

284
00:25:18,000 --> 00:25:23,000
is highly condensed.
I've told you previously that it's

285
00:25:23,000 --> 00:25:27,000
a real feat to pack the meter of DNA
that's in every cell,

286
00:25:27,000 --> 00:25:31,000
into a little, tiny nucleus.
You have to really pack it tightly.

287
00:25:31,000 --> 00:25:35,000
Well, it's even worse for a sperm
because the nucleus of a sperm is an

288
00:25:35,000 --> 00:25:40,000
order of magnitude smaller,
in many cases, than a somatic cell.

289
00:25:40,000 --> 00:25:44,000
So you have to pack the DNA even
more tightly, and there are special

290
00:25:44,000 --> 00:25:48,000
proteins that overcome the charges
on the DNA, to overcome the charge

291
00:25:48,000 --> 00:25:52,000
repulsion on the deoxyribonucleic
acid, OK?  So these are basic

292
00:25:52,000 --> 00:25:57,000
proteins that really pack the DNA
tightly, to fit into this compact

293
00:25:57,000 --> 00:26:01,000
nucleus.  The other thing we'll talk
about, in a bit,

294
00:26:01,000 --> 00:26:06,000
is this structure called the
acrosome.

295
00:26:06,000 --> 00:26:10,000
It derives from the Golgi,
which you remember is involved in

296
00:26:10,000 --> 00:26:14,000
protein trafficking.
Two other regions of the sperm that

297
00:26:14,000 --> 00:26:18,000
are almost universal,
and very important, are this thing

298
00:26:18,000 --> 00:26:22,000
called the mid-piece,
which is packed with mitochondria

299
00:26:22,000 --> 00:26:26,000
that are going to provide energy for
the sperm as it moves.

300
00:26:26,000 --> 00:26:30,000
And finally, this tail region,
which is a flagellum, now what's a

301
00:26:30,000 --> 00:26:35,000
flagellum?
So, a flagellum is one of these

302
00:26:35,000 --> 00:26:39,000
biologically fantastic structures
that uses polymers of proteins to

303
00:26:39,000 --> 00:26:44,000
make something that works in a
concerted fashion.

304
00:26:44,000 --> 00:26:48,000
The proteins that it uses makes up
the microtubules,

305
00:26:48,000 --> 00:26:52,000
so they are the tubulin proteins,
we'll have more to say about them on

306
00:26:52,000 --> 00:26:57,000
Friday.  We mentioned microtubules
in the last lecture,

307
00:26:57,000 --> 00:27:01,000
in this case, microtubules pack
together in a very ordered way,

308
00:27:01,000 --> 00:27:07,000
you can read this in your book.
Microtubules pack together in a very

309
00:27:07,000 --> 00:27:13,000
ordered way, to make this very long
structure, that when supplied with

310
00:27:13,000 --> 00:27:20,000
energy, will beat in a regular,
often a sinusoidal fashion, and

311
00:27:20,000 --> 00:27:26,000
propel the cell forward,
or wherever it is going.  All right,

312
00:27:26,000 --> 00:27:32,000
female reproductive system.
The ovary is the sight of egg

313
00:27:32,000 --> 00:27:37,000
production.  In mammals,
the uterus is the site of embryo

314
00:27:37,000 --> 00:27:42,000
growth, fertilization usually occurs
somewhere between the ovary and the

315
00:27:42,000 --> 00:27:47,000
uterus, in the tube,
the oviduct, or the fallopian tube,

316
00:27:47,000 --> 00:27:56,000
between the ovary and the uterus.

317
00:27:56,000 --> 00:28:00,000
All right, this is a diagram from
your book again.

318
00:28:00,000 --> 00:28:04,000
In the developing,
in the ovary, one can find eggs of

319
00:28:04,000 --> 00:28:08,000
developing, or oocytes sites of
different stages,

320
00:28:08,000 --> 00:28:12,000
as they undergo development.
They grow larger, and lie in a

321
00:28:12,000 --> 00:28:16,000
fluid-filled cavity,
and eventually, I'll talk a bit more

322
00:28:16,000 --> 00:28:20,000
about this in a moment,
this fluid filled cavity gets very

323
00:28:20,000 --> 00:28:24,000
large, and it bursts, and
releases the egg.

324
00:28:24,000 --> 00:28:28,000
It's very interesting,
in mammals, the egg is actually

325
00:28:28,000 --> 00:28:33,000
released almost into the body cavity,
and it's gathered up by so called

326
00:28:33,000 --> 00:28:38,000
fimbrae, or folds of waving tissue
that lie at the opening of the

327
00:28:38,000 --> 00:28:42,000
oviduct, or fallopian tube,
and these kind of make a current

328
00:28:42,000 --> 00:28:47,000
that draws the released egg into the
oviduct, so it can get on its way to

329
00:28:47,000 --> 00:28:51,000
the uterus.
Subsequent  to the egg being

330
00:28:51,000 --> 00:28:55,000
released, what's left of where the
egg developed in the ovary,

331
00:28:55,000 --> 00:28:58,000
forms this thing called the corpus
luteum, which secretes substances,

332
00:28:58,000 --> 00:29:02,000
hormones, that I'll talk about in a
moment, that are very important for

333
00:29:02,000 --> 00:29:06,000
maintaining pregnancy,
if this occurs.

334
00:29:06,000 --> 00:29:11,000
All right, you can see what I'm
dwelling on here,

335
00:29:11,000 --> 00:29:16,000
not dwelling on.  I'll dwell on
things that I feel are more

336
00:29:16,000 --> 00:29:21,000
important, by writing things on the
board, OK?  So,

337
00:29:21,000 --> 00:29:26,000
stay with me.  Let's go now to a
system that I do want to dwell on

338
00:29:26,000 --> 00:29:31,000
more, which is the hormonal control
of gametogenesis.

339
00:29:31,000 --> 00:29:36,000
Professor Jacks talked to you
previously about hormones.

340
00:29:36,000 --> 00:29:47,000
Hormones are made in most animals,

341
00:29:47,000 --> 00:29:53,000
and their characteristic is that
they work at very long range.

342
00:29:53,000 --> 00:29:59,000
So I want to characterize these as
long-ranged, inducers,

343
00:29:59,000 --> 00:30:06,000
and they can be very long-range
inducers.  The hormones that control

344
00:30:06,000 --> 00:30:12,000
gametogenesis are made in the brain,
in the hypothalamus, and the

345
00:30:12,000 --> 00:30:18,000
pituitary and they travel way
through the bloodstream to get to

346
00:30:18,000 --> 00:30:24,000
the ovaries and the testes.
This looks like a hermaphrodite here,

347
00:30:24,000 --> 00:30:29,000
[LAUGHTER], OK and this,
I just realized this, OK,

348
00:30:29,000 --> 00:30:34,000
whatever.  To get to the ovaries or
the testes, or both,

349
00:30:34,000 --> 00:30:39,000
whatever, {LAUGHTER],
OK, anyway, that point I want to

350
00:30:39,000 --> 00:30:44,000
make is that these hormones work at
a very long distance.

351
00:30:44,000 --> 00:30:49,000
And I want to contrast that with
the kinds of inducers that wee have

352
00:30:49,000 --> 00:30:54,000
been talking about in the preceding
couple of lectures.

353
00:30:54,000 --> 00:30:59,000
The kind of classical,
embryonic inducers, which act at

354
00:30:59,000 --> 00:31:05,000
very short range.
So, for example, we talked about

355
00:31:05,000 --> 00:31:14,000
nodal signaling --

356
00:31:14,000 --> 00:31:18,000
-- last lecture.
For example, nodal,

357
00:31:18,000 --> 00:31:22,000
which is a ligand I mentioned,
pivotal in making the mesoderm,

358
00:31:22,000 --> 00:31:26,000
nodal can only work over one, or a
few, cell diameters.

359
00:31:26,000 --> 00:31:30,000
So if you were cells,
you could have a conversation with

360
00:31:30,000 --> 00:31:34,000
one another, or maybe,
the signal could get all the way to

361
00:31:34,000 --> 00:31:38,000
here, but it sure wouldn't get to
the back of the room.

362
00:31:38,000 --> 00:31:44,000
Whereas, if you were a hormone,
your signal would get all the way

363
00:31:44,000 --> 00:31:50,000
back to the back of the room,
and probably all the way down the

364
00:31:50,000 --> 00:31:56,000
infinite corridor.
OK, so there's a real order of

365
00:31:56,000 --> 00:32:02,000
magnitude different in the way
hormones and other inducers can act.

366
00:32:02,000 --> 00:32:08,000
So, one to a few cells.
All right, there are a bunch of

367
00:32:08,000 --> 00:32:13,000
different kinds of hormones that are
involved in control of gametogenesis.

368
00:32:13,000 --> 00:32:18,000
I want to distinguish the steroid
hormones, which are derivatives of

369
00:32:18,000 --> 00:32:23,000
cholesterol, and we mentioned them
long ago in the lipid section.

370
00:32:23,000 --> 00:32:28,000
Steroids that include testosterone,
estrogen, and progesterone, and act

371
00:32:28,000 --> 00:32:33,000
through a series of receptors that
actually lies within the cell,

372
00:32:33,000 --> 00:32:38,000
they're not membrane-bound receptors,
they are so-called nuclear receptors,

373
00:32:38,000 --> 00:32:44,000
so they act through
nuclear receptors.

374
00:32:44,000 --> 00:32:48,000
I'm actually not going to write that
on the board.  And the other

375
00:32:48,000 --> 00:32:53,000
hormones I want to indicate are
peptide hormones,

376
00:32:53,000 --> 00:32:57,000
that as their name indicates,
are small polypeptide chains, and

377
00:32:57,000 --> 00:33:02,000
these include,
I'm going to put abbreviations up,

378
00:33:02,000 --> 00:33:07,000
and you can get the full names later.
FSH, LH, and GNRH,

379
00:33:07,000 --> 00:33:11,000
follicle-stimulating hormone,
luteinizing hormone, and

380
00:33:11,000 --> 00:33:16,000
gonadotropin-releasing hormone.
One of the things that is similar

381
00:33:16,000 --> 00:33:20,000
about the hormonal control of
gametogenesis,

382
00:33:20,000 --> 00:33:25,000
to many other things we've been
talking about,

383
00:33:25,000 --> 00:33:30,000
is the notion of feedback control.
So, there is an enormous amount of

384
00:33:30,000 --> 00:33:34,000
feedback, both positive and negative
in the circuitry that gives rise to

385
00:33:34,000 --> 00:33:39,000
the formation of egg and sperm.
This is an example of the circuitry

386
00:33:39,000 --> 00:33:44,000
leading to stomatogenesis.
Here is GNRH, released from the

387
00:33:44,000 --> 00:33:49,000
hypothalamus that stimulates the
anterior pituitary,

388
00:33:49,000 --> 00:33:54,000
to produce lutenizing hormone,
or follicle stimulating hormone.

389
00:33:54,000 --> 00:34:00,000
These act on cells within the
testes, which act to,

390
00:34:00,000 --> 00:34:05,000
one set of cells acts to activate
testosterone, the major male steroid

391
00:34:05,000 --> 00:34:10,000
hormone, and that acts back on the
Sertoli cells in the seminiferous

392
00:34:10,000 --> 00:34:15,000
tubules, and together,
these orchestrate the production of

393
00:34:15,000 --> 00:34:21,000
spermatozoa, and other various
responses to these hormones.

394
00:34:21,000 --> 00:34:26,000
Now, the green indicates positive
loops, and the red indicate negative

395
00:34:26,000 --> 00:34:32,000
feedback, so too much testosterone
can have the effect of inhibiting

396
00:34:32,000 --> 00:34:38,000
the anterior pituitary's production
of lutenizing hormone,

397
00:34:38,000 --> 00:34:44,000
or follicle-stimulating hormone.
And there's a very careful balance

398
00:34:44,000 --> 00:34:50,000
of these hormones in the normal
animal.  Now, in females,

399
00:34:50,000 --> 00:34:56,000
things look similar.  Again,
GNRG, LH, and FSH, are produced to

400
00:34:56,000 --> 00:35:02,000
stimulate, release,
and maturation of oocytes,

401
00:35:02,000 --> 00:35:07,000
and release from the ovary.
The ovary, instead of producing

402
00:35:07,000 --> 00:35:12,000
testosterone, although it does
produce some of that,

403
00:35:12,000 --> 00:35:16,000
produces estrogen and progesterone,
and those are both required for

404
00:35:16,000 --> 00:35:21,000
normal maturation of the egg,
and also for subsequent pregnancy.

405
00:35:21,000 --> 00:35:26,000
And also, as you will see, there is
a feedback loop of both estrogen and

406
00:35:26,000 --> 00:35:31,000
progesterone, onto the hypothalamus
and the anterior pituitary.

407
00:35:31,000 --> 00:35:34,000
And as this feedback loop that is
exploited in the birth control pill,

408
00:35:34,000 --> 00:35:38,000
where if one gives high levels of
estrogen and progesterone,

409
00:35:38,000 --> 00:35:42,000
you suppress the production of both
GNRH from the hypothalamus,

410
00:35:42,000 --> 00:35:46,000
and LH and FSH from the anterior
pituitary and therefore suppress the

411
00:35:46,000 --> 00:35:50,000
production of eggs from the ovary.
Now actually looking, it's

412
00:35:50,000 --> 00:35:54,000
interesting that there's a female
birth control pill,

413
00:35:54,000 --> 00:35:58,000
because really, there are certainly
targets in the male for birth

414
00:35:58,000 --> 00:36:02,000
control pills,
but these have not yet been

415
00:36:02,000 --> 00:36:06,000
exploited.
But the notion is that you exploit

416
00:36:06,000 --> 00:36:11,000
this feedback loop,
and its well-enough understood that

417
00:36:11,000 --> 00:36:17,000
you can.  OK, so let me move on to
talking about the process of

418
00:36:17,000 --> 00:36:24,000
fertilization, per say.

419
00:36:24,000 --> 00:36:28,000
The process of fertilization
involves the egg and the sperm

420
00:36:28,000 --> 00:36:32,000
coming together,
and recognizing one another as being

421
00:36:32,000 --> 00:36:37,000
from the same species.  This
is very important --

422
00:36:37,000 --> 00:36:46,000
-- very important that you get

423
00:36:46,000 --> 00:36:50,000
species-specific recognition,
in order that you get correct

424
00:36:50,000 --> 00:36:54,000
transmission of genetic material.
So the first thing that we mention

425
00:36:54,000 --> 00:37:03,000
is sperm-egg recognition --

426
00:37:03,000 --> 00:37:07,000
-- and this has many levels.
In some animals, the egg, or the

427
00:37:07,000 --> 00:37:12,000
uterus, or the female sends out
various chemo attractants to

428
00:37:12,000 --> 00:37:17,000
attractants, to encourage the sperm
to get to the egg.

429
00:37:17,000 --> 00:37:22,000
Some of these have been isolated,
and there are small peptides, for

430
00:37:22,000 --> 00:37:27,000
example, sea urchins,
which are the organism that have

431
00:37:27,000 --> 00:37:31,000
been used to study fertilization.
Most have very small peptides,

432
00:37:31,000 --> 00:37:35,000
or the chemo attractants, in humans
there is some data that there may be

433
00:37:35,000 --> 00:37:39,000
chemo attractants released by the
oviduct that causes the sperm to

434
00:37:39,000 --> 00:37:43,000
swim in a directed fashion,
but it's very interesting that they

435
00:37:43,000 --> 00:37:47,000
can't be that powerful,
those chemo attractants,

436
00:37:47,000 --> 00:37:50,000
because one needs so many sperm to
be released in order to fertilize

437
00:37:50,000 --> 00:37:54,000
the egg.  And it's very interesting
that although there are five times

438
00:37:54,000 --> 00:37:58,000
ten to the seventh,
or more, spermatozoa in a human

439
00:37:58,000 --> 00:38:02,000
ejaculate, if you actually look in
the region of the oviduct,

440
00:38:02,000 --> 00:38:06,000
where fertilization would take place,
you only find a few hundred

441
00:38:06,000 --> 00:38:10,000
sperm there.
So most of them disappear on the way,

442
00:38:10,000 --> 00:38:14,000
and so if there are chemo
attractants, I say they're not very

443
00:38:14,000 --> 00:38:18,000
good ones.  OK,
another process that is very

444
00:38:18,000 --> 00:38:23,000
important in mammals,
is something called capacitation.

445
00:38:23,000 --> 00:38:27,000
The sperm, as I told you, have this
capacity to be motile,

446
00:38:27,000 --> 00:38:31,000
that they contain a lot of energy
stores, or they contain mitochondria

447
00:38:31,000 --> 00:38:36,000
that can convert energy
to movement.

448
00:38:36,000 --> 00:38:42,000
But they save that energy for a time
when they think they might be in the

449
00:38:42,000 --> 00:38:48,000
vicinity of an egg,
and when sperm are exposed to fluid

450
00:38:48,000 --> 00:38:54,000
from a mammalian oviduct,
they undergo an increase in motility,

451
00:38:54,000 --> 00:39:01,000
so capacitation leads to an increase
in sperm motility.

452
00:39:01,000 --> 00:39:07,000
This process is mediated by some
kind of signal transduction

453
00:39:07,000 --> 00:39:13,000
mechanism, that involves the second
messenger, cyclic AMP that you

454
00:39:13,000 --> 00:39:20,000
discussed with Professor Jacks,
and this is very important.

455
00:39:20,000 --> 00:39:24,000
If capacitation doesn't occur,
fertilization doesn't occur either.

456
00:39:24,000 --> 00:39:29,000
OK, so once the sperm are in the
vicinity of the egg,

457
00:39:29,000 --> 00:39:34,000
they then have the challenge of
fusing with it.

458
00:39:34,000 --> 00:39:38,000
This is an animation I showed you
previously, of a sperm moving

459
00:39:38,000 --> 00:39:43,000
towards an egg.
Now this is a mammalian egg,

460
00:39:43,000 --> 00:39:48,000
and it's surrounded by a bunch of
stuff.  It's surrounded by these

461
00:39:48,000 --> 00:39:52,000
circly things,
which are cells that released from

462
00:39:52,000 --> 00:39:57,000
the ovary, along with the egg,
and it's surrounded by a number of

463
00:39:57,000 --> 00:40:01,000
so-called membranes.
The most important one of which is

464
00:40:01,000 --> 00:40:05,000
called the zona pellucida,
and as the sperm goes towards the

465
00:40:05,000 --> 00:40:09,000
egg, and approaches the egg,
it has to burrow its way through a

466
00:40:09,000 --> 00:40:13,000
bunch of membranes to get there,
this animation doesn't finish

467
00:40:13,000 --> 00:40:16,000
fertilization clearly.
OK, so if you look at a mammalian

468
00:40:16,000 --> 00:40:20,000
egg at fertilization,
there is the egg per-say,

469
00:40:20,000 --> 00:40:24,000
that is a cell surrounded by a
plasma membrane,

470
00:40:24,000 --> 00:40:28,000
and then there's this thick,
yellow layer, that's called the zona

471
00:40:28,000 --> 00:40:32,000
pellucida.
Around that, is this layer of cells

472
00:40:32,000 --> 00:40:37,000
called the cumulus,
and the sperm have to borrow through

473
00:40:37,000 --> 00:40:42,000
both the cumulus layer,
and particularly the zona pellucida,

474
00:40:42,000 --> 00:40:47,000
before they get to the plasma
membrane, and before they can then

475
00:40:47,000 --> 00:40:52,000
try to fuse with the plasma membrane,
and enter the egg.

476
00:40:52,000 --> 00:40:57,000
So, the second thing that we need
to think about is contact,

477
00:40:57,000 --> 00:41:02,000
and there's two steps to this.
The first contact and they're

478
00:41:02,000 --> 00:41:07,000
actually very interesting steps
because there's a bunch of

479
00:41:07,000 --> 00:41:13,000
reciprocal interaction,
and reciprocal receptor ligand

480
00:41:13,000 --> 00:41:18,000
interaction.  The first interaction
is between the sperm,

481
00:41:18,000 --> 00:41:23,000
which provides a receptor,
and the egg, and this layer called

482
00:41:23,000 --> 00:41:29,000
the zona pellucida around the egg,
and the zona provides the ligands

483
00:41:29,000 --> 00:41:34,000
for the interaction.
And it turns out that the zona

484
00:41:34,000 --> 00:41:40,000
pellucida, although it's called a
membrane very often,

485
00:41:40,000 --> 00:41:46,000
is not a lipid by-layer,
it's a glycol-protein layer,

486
00:41:46,000 --> 00:41:51,000
OK?  And the proteins in the zona
are called zp1,

487
00:41:51,000 --> 00:41:57,000
zp2, and zp3, and they form a very
complex meshwork of

488
00:41:57,000 --> 00:42:02,000
glycol-proteins.
The sperm receptor in mammals

489
00:42:02,000 --> 00:42:06,000
recognizes a protein called zp3 in
the zona, and then subsequently,

490
00:42:06,000 --> 00:42:11,000
there's a shift in carbohydrate
arrangement of the zona proteins,

491
00:42:11,000 --> 00:42:16,000
and it then recognizes something
called zp2.  Now,

492
00:42:16,000 --> 00:42:20,000
this receptor-ligand interaction has
a very interesting consequence.

493
00:42:20,000 --> 00:42:25,000
It leads to activation of second
messengers, it goes through a system

494
00:42:25,000 --> 00:42:30,000
of proteins called G proteins,
but what was most important is that

495
00:42:30,000 --> 00:42:35,000
it leads to the release of calcium
within the sperm.

496
00:42:35,000 --> 00:42:41,000
And this calcium,
let me move the calcium here,

497
00:42:41,000 --> 00:42:47,000
so this leads to the release of
calcium with the sperm,

498
00:42:47,000 --> 00:42:53,000
and this causes something called the
acrosome reaction.

499
00:42:53,000 --> 00:42:59,000
Now the acrosome, as I said,
derives from the Golgi, and it is

500
00:42:59,000 --> 00:43:06,000
filled with Golgi-derived proteins,
particularly proteases.

501
00:43:06,000 --> 00:43:11,000
Upon the acrosome reaction,
there is a massive exocytosis of the

502
00:43:11,000 --> 00:43:17,000
acrosomal contents,
to release proteases,

503
00:43:17,000 --> 00:43:23,000
and these proteases digest the zona.
All right, some diagrams.  This is

504
00:43:23,000 --> 00:43:28,000
a diagram you have on your handout.
Here is the sperm moving through

505
00:43:28,000 --> 00:43:34,000
the cumulus layer,
getting through the zona,

506
00:43:34,000 --> 00:43:40,000
at contact there is a reaction,
such that the acrosome releases its

507
00:43:40,000 --> 00:43:46,000
contents, and the next
step can happen.

508
00:43:46,000 --> 00:43:51,000
This is a movie of the acrosome
reaction from the horseshoe crab,

509
00:43:51,000 --> 00:43:56,000
limulus, it has an extraordinary
acrosome.  The acrosome in limulus

510
00:43:56,000 --> 00:44:01,000
compromises acrosomal protein,
the proteases, but a lot of

511
00:44:01,000 --> 00:44:06,000
unpolymerized,
and upon contact with the limulus

512
00:44:06,000 --> 00:44:11,000
egg, the, let me stop this so you
can watch it while I'm telling

513
00:44:11,000 --> 00:44:16,000
you about it.
The actin that is in the acrosome

514
00:44:16,000 --> 00:44:21,000
polymerizes, this is a matter of
seconds, and sends out this

515
00:44:21,000 --> 00:44:26,000
incredibly long process,
here it comes, look at this.

516
00:44:26,000 --> 00:44:30,000
See this thing shooting out?
That is a polymer of actin that is

517
00:44:30,000 --> 00:44:35,000
coated with these acrosomal
proteases.  So this is the sperm

518
00:44:35,000 --> 00:44:40,000
head, and so it's shooting out this
process that is many,

519
00:44:40,000 --> 00:44:45,000
many times longer than the sperm
head, and it's allowing it to get

520
00:44:45,000 --> 00:44:50,000
into the limulus egg.
Mammalian sperm do not have such a

521
00:44:50,000 --> 00:44:55,000
spectacular acrosome reaction,
but they do have one that serves the

522
00:44:55,000 --> 00:45:00,000
same purpose.  OK,
all right, so the next thing that is

523
00:45:00,000 --> 00:45:05,000
going to happen is that there is a
second reciprocal interaction,

524
00:45:05,000 --> 00:45:10,000
and this time, the egg is going to
provide the receptor,

525
00:45:10,000 --> 00:45:16,000
and the sperm is going to provide
the ligand.

526
00:45:16,000 --> 00:45:22,000
So, we now have the sperm,
whose acrosomal membrane that had

527
00:45:22,000 --> 00:45:29,000
been the Golgi membrane,
is now exposed, and is covered with

528
00:45:29,000 --> 00:45:36,000
ligands, so the acrosome,
the acrosome membrane, is covered

529
00:45:36,000 --> 00:45:42,000
with ligand.
This interacts with receptors on the

530
00:45:42,000 --> 00:45:48,000
egg plasma membrane,
and there is, as a result of this

531
00:45:48,000 --> 00:45:54,000
receptor-ligand interaction,
the activation of a

532
00:45:54,000 --> 00:46:00,000
signal-transduction system,
and the signal transduction system

533
00:46:00,000 --> 00:46:05,000
does two things,
so there is this interaction leads

534
00:46:05,000 --> 00:46:11,000
within the egg,
to activation of signal transduction,

535
00:46:11,000 --> 00:46:17,000
that has the effect,
eventually, of releasing calcium

536
00:46:17,000 --> 00:46:23,000
with the egg.  And this calcium
released within the egg

537
00:46:23,000 --> 00:46:28,000
does two things.
The first thing that it does is to

538
00:46:28,000 --> 00:46:33,000
prevent any additional sperm from
entering the egg,

539
00:46:33,000 --> 00:46:38,000
and I'll tell you about this in a
second.  So it blocks,

540
00:46:38,000 --> 00:46:43,000
or activates the block,
to polyspermy, and in particular it

541
00:46:43,000 --> 00:46:48,000
activates the so-called slow block
to polyspermy,

542
00:46:48,000 --> 00:46:53,000
and it also activates,
a little while later, the zygote,

543
00:46:53,000 --> 00:46:58,000
to begin dividing, to enter the cell
cycle, and to begin various other

544
00:46:58,000 --> 00:47:04,000
processes required in
the early embryo.

545
00:47:04,000 --> 00:47:10,000
So cell cycle entry of the zygote.
All right, so let's look at a movie

546
00:47:10,000 --> 00:47:17,000
of the sperm entering the egg.
OK, here is, I'll show you this

547
00:47:17,000 --> 00:47:23,000
again.  Here is the sperm,
its tale is sticking out, there,

548
00:47:23,000 --> 00:47:30,000
it's going right into the egg.  The
cell membrane reciprocates,

549
00:47:30,000 --> 00:47:36,000
and buckles, and in some organisms,
it engulfs the sperm as it's going

550
00:47:36,000 --> 00:47:43,000
in, but the actual sense in most
organisms, the membranes either fuse,

551
00:47:43,000 --> 00:47:50,000
or somehow you get the whole sperm
being pushed into the egg.

552
00:47:50,000 --> 00:47:55,000
In most animals the sperm membrane
doesn't actually enter the egg,

553
00:47:55,000 --> 00:48:01,000
but in some animals it does, and in
some cases it doesn't seem to matter.

554
00:48:01,000 --> 00:48:07,000
OK, what is this block to
polyspermy?  This is something that

555
00:48:07,000 --> 00:48:13,000
happens in the first 30 seconds
after fertilization,

556
00:48:13,000 --> 00:48:19,000
and it involves exocytosis of a set
of granules called the cortical

557
00:48:19,000 --> 00:48:25,000
granules, that contain enzymes,
that do two things.

558
00:48:25,000 --> 00:48:28,000
These enzymes firstly,
when they are released,

559
00:48:28,000 --> 00:48:32,000
break apart junctions between the
outer membranes,

560
00:48:32,000 --> 00:48:36,000
in this case, this is shown in frogs.
The outer membrane in frogs is

561
00:48:36,000 --> 00:48:40,000
called the vitelline envelope,
its equivalent to the zona pellucida

562
00:48:40,000 --> 00:48:44,000
in mammals.  And before
fertilization,

563
00:48:44,000 --> 00:48:48,000
this outer envelope is tethered by
proteins, to the plasma membrane,

564
00:48:48,000 --> 00:48:52,000
so they're very closely opposed.
At the release of the cortical

565
00:48:52,000 --> 00:48:56,000
granules, these protein contacts are
broken, and the fertilization

566
00:48:56,000 --> 00:49:00,000
envelope lifts off the surface of
the egg.  A number of other things

567
00:49:00,000 --> 00:49:05,000
happen, as well.
In mammals the zona pellucida is

568
00:49:05,000 --> 00:49:09,000
completely remodeled,
so it no longer binds with the sperm,

569
00:49:09,000 --> 00:49:13,000
the carbohydrates are stripped off
the zona proteins so that they no

570
00:49:13,000 --> 00:49:18,000
longer bind sperm.
This is a movie of the block to

571
00:49:18,000 --> 00:49:23,000
polyspermy, or the consequence of
the block to polyspermy in the frog.

572
00:49:23,000 --> 00:49:28,000
Here is the frog egg.  At
fertilization,

573
00:49:28,000 --> 00:49:33,000
watch carefully,
you can see there is a big halo that

574
00:49:33,000 --> 00:49:38,000
arises above the frog egg,
there it is, and this is a

575
00:49:38,000 --> 00:49:43,000
consequence of cortical granule
exocytosis, and the blocked

576
00:49:43,000 --> 00:49:48,000
polyspermy.
OK.  OK.  Before you go,

577
00:49:48,000 --> 00:49:51,000
what I did not cover in lecture
today, don't go for one second.