Re: [Histonet] shark egg carbohydrate jelly staining

From:John Kiernan

The sugars you name in the first paragraph are ones
that go with glycoproteins. Even though acidic sugars
were not mentioned in the "only one paper," they
must be present in the components separated by
electrophoresis, or alcian blue staining would not
occur, because this dye stains only sugar-acids.

The change to opaque white in formaldehyde indicates 
that protein is present because formaldehyde does not
react chemically with most carbohydrates; it does
cross-link and insolubilize proteins. Your eggs
are quite large specimens, and it's difficult to
imagine a dye penetrating evenly to the centre and
forming a meaningful gradient of colour. If you do
not have the equipment or expertise to make sections
your best bet may be to collaborate with someone
who has. Marine biology labs must surely do that
kind of work.
John A. Kiernan
Department of Anatomy and Cell Biology
The University of Western Ontario
London,   Canada   N6A 5C1
Jen Wyffels & Greg Shafer wrote:
> Dr. Kiernan and other histonet readers,
> More information about the egg case jelly in sharks:
> There has been only one paper that investigated the composition of the 3
> different egg jellys in elasmobranch eggs.  In that report there was no
> protein associated with any jelly layer.  The different layers were analyzed
> after hydrolysis for neutral and amino sugars by HPLC and found to contain
> different proportions and increasing concentrations of the same 4 sugars,
> glucosamine, galactose, galactosamine and fucose.  Note, acidic sugars if
> present were not measured.  The liquid egg jelly (1) was analyzed by gel
> electrophoresis and had components ranging in size from 31 to 200 kDa as
> stained by alcian blue, and again no protein was found in the same samples
> (comasssie blue stain).
> Size does matter....these eggs are at the smallest 70mm by 20 mm, 15mm thick
> and the larger eggs are 150mm by 70mm, 35mm thick, both measurements include
> the egg shell (0.5mm thick).  I am afraid I cannot embed and section either
> egg.  I was thinking more of immersing the entire egg after removing the top
> half of the shell to expose the contents within.  I was hoping that the
> jelly layers would stain differently (either a single stain or multiple) and
> yield a gradient of color(s) from the center liquid jelly (1) through the
> next mucus like jelly (2) to the edges of the egg case where the dense jelly
> (3) is concentrated.  I can easily use a fixative on the entire egg, but
> complete penetration through the jelly may not be possible without
> disrupting its normal distribution.  The dense jelly 3 will not separate
> from the egg case but the mucus jelly (2) and the liquid jelly (1) are
> readily lost if the egg is tipped over.  I realize the liquid jelly (1) will
> be lost in any staining technique so I will remove it and attempt to stain
> it with the same method as the egg but in a test tube.  I have already
> prepared some eggs with embryos in 10% formalin and I can add that the jelly
> (2) turns from a clear snot to an opaque white color after fixation.  The
> dense jelly (3) is opaque and sometimes has a slight yellow cast before
> fixation.
> I hope this information helps, I appreciate the time you have taken to
> consider this staining adventure.  Jen
> ----- Original Message -----
> From: "John Kiernan" 
> To: "Jen Wyffels & Greg Shafer" 
> Cc: 
> Sent: Sunday, October 24, 2004 1:11 PM
> Subject: Re: [Histonet] shark egg carbohydrate jelly staining
> > If the biochemistry of shark egg jelly is known
> > (as you indicate), it will probably be possible to
> > localize different carbohydrate components in
> > different colours. Are the carbohydrates bound to
> > protein, as glycoproteins or proteoglycans? If so,
> > they will be fixable with formaldehyde and you will
> > be able to do the staining the usual way - on sections
> > rather than whole specimens. Even if the jelly is
> > protein-free it must consist of very large polysacharide
> > molecules at least in the mucous and solid layers, and
> > can probably insolubilized by a suitable fixative.
> >
> > The choice of staining methods for carbohydrates is
> > quite large, and it's possible to collect significant
> > chemical information. These techniques have all
> > been developed for sections (usually paraffin). Some
> > of the simpler techniques can be applied to very small
> > (0.5 mm) whole objects that are later embedded and
> > sectioned for electron microscopy.
> >
> > How big is a shark's egg? That affects the technology!
> > If you can provide more biochemical information about
> > shark egg jelly, I (and others reading Histonet
> > messages) will be able to advise you about preparative
> > techniques and staining methods.
> >
> > John A. Kiernan
> > Dept of Anatomy & Cell Biology
> > University of Western Ontario
> > London,  Canada  N6A 5C1
> > _____________________________________________
> > Jen Wyffels & Greg Shafer wrote:
> > >
> > > I would like to stain the carbohydrate jelly in a shark egg.  The egg is
> > > similar to a chicken egg, the yolk is surrounded by a protective matrix
> but
> > > in sharks it is carbohydrate rather than albumen.  I will remove the
> yolk
> > > leaving the egg jelly within the egg shell.  This jelly has 3 distinct
> and
> > > separate compositions, a water like layer (1), a 'snot' like layer (2)
> and
> > > finally a very dense solid (3).  I would like to immerse this egg in a
> > > carbohydrate stain that would show the layers and their distribution
> withing
> > > the egg case.  These layers have been shown (after complete hydrolysis)
> to
> > > differ in the concentration of sugar moeties present with the dense
> layer
> > > containg the highest concentrations.  Has anyone tried something similar
> and
> > > willing to share their results?  I can experiment a little but egg
> > > availability is limited so any advice is welcome.  Thanks,  Jen
> > >
> > > _______________________________________________
> > > Histonet mailing list
> > >
> > >

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