I have produced some embryos that contain cells without nucleii.
dear vahid, certainly it is a very confusing questions, because if you have morula and can not see nucleii insider the cells, you are not in fronto of a morula, more likely you are in front of a degenerated or fragmented embryo or oocyte. this is the reasonf of DNA dispearing.
As Jesus outlined, the most likely explanation is that the embryos exhibit irregular fragmentation events not preceded by a mitosis.
What do you think about this picture? why inside of some cells are empty? which staining you suggest to understand more?
Dear Vahid, so wich are your question,, I see 15 nuclei, how DNA you are expecting to get? how many cells are you seeing in that morulae?
Dear Vahid,
I suppose the blue staining is DAPI. You could try Propidium Iodide staining to check for single stranded DNA.
Did you consider the possibility your blue laser or light source does not reach the far end of your embryo and the 'lack of staining' is an artefact of this?
this picture in viewing by light microscope is morula. My question is empty parts of the murola that there is no nuclei while we can see walls of the cells. Why in my staining by Hoechst and ROS I see empty parts while must be filled by nuclei?
lack of staining can happen but why is same for all embryos?
Dear Vahid,
If it is indeed a staining or imaging artefact it will definitely happen in all the embryos. Sometimes the penetration of the light (or the antibody) is not the same for all colours, and I know that a morula can be pretty dense. How do you permeabilize?
But also try another DNA staining, that can detect single stranded DNA, like PI.
Please let us know what you find out!
I am writing a proposal to study more about these occurrences. I will use Annexin V and Hoechst and if I could i will try gel electrophoresis. Of course gene expression will be useful too but I do not have grant for them. :O(
if you have grant we can design some experiments to investigate it. :O)
Dear Vahid, if you post a transmission light picture of your embryo, that would help understand better the shape and the size of the blastomeres with empty nuclei.
Thank you,
Arturo
Dear Vahid, don't you have a phase contrast image of the embryo?
Thank you,
Arturo
The problem with fluorescent light images is that you don't see the shape of the embryo or of the blastomeres. If your embryo is a morula, the absence of stained nuclei would be a sign of abnormality. but if you are looking at an early blastocyst, you may see flat blastomeres with periferic nuclei and the embryo would still be perfectly normal. In your case, you should compare the number of blastomeres observed in phase contrast with the number of nuclei stained with Hoechst. So, please try to locate other images of your embryos.
Arturo
i am sure that they are morula as i have checked on more than 1000 embryos.
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