Re: [Histonet] shark egg carbohydrate jelly staining

From:"Jen Wyffels & Greg Shafer"

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
> > Histonet@lists.utsouthwestern.edu
> > http://lists.utsouthwestern.edu/mailman/listinfo/histonet


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