I know that there is a correlation between cumulus expention of Cumulus Oocyte Complexes (COCs) and nuclear maturation of oocyte in in vitro maturation (IVM). However, it is well known that cumulus expansion is not directly correlated with nuclear maturation. So it is recommended to stain with aceto-orcein to determine the exact phase of nuclear material of oocyte (MII plate formation or Germinal Vesicle(GV) break down). The staining causes the dead of oocyte as you know and you can not use the oocyte for In Vitro Fertilization processes. Do you use first polar body formation to determine maturation of oocyte after IVM? But I know it is difficult to determine first polar body in all COCs or denuded oocytes. Or do you know any vital staining or method to determine oocyte maturation (MII plate formation or GV breakdown)?
Dear Umut,
If you are going to do IVF, so you should use the COCs and it is not recommended to denude the oocyte to determine the GVBD or 1st polar body. The popular indicator is the cumulus expansion.
But if you are going to do ICSI, so you should denude the oocyte and judge the maturation by 1st polar body extrusion using stereomicroscope or through the micromanipulator attached to the inverted microscope during ICSI procedures, you can observe the 1st PB more precisely with no harm to the oocytes.
Dear Islam,
Thank you for your answer. Some reviewers can not accept cumulus expension as a indicator of maturation. They want aceto-orcein staining for sample of COCs after IVM. However, as you know IVF is a continuing process including IVM, IVF and IVC. The process of aceto-orcein staining is not vital staining. Do you know any vital staining for determination of GVBD or MII plate formation after IVM?
The safer method is Hoechst stain. Fully denuded oocytes will be subjected to 2 microM of Hoechst stain in TALP solution for 5 min. You can observe 2 blue fluorescence spots (the nucleus and 1st PB) after brief exposure to UV rays. You can wash the oocytes several times after that using TALP.
According to my experience, you may have adverse outcomes because of the staining and the UV rays.
Dear Umut
I think you may misunderstand about the requirement of reviewers. Probably, they want you to check maturation rate of oocyte in your system by aceto-orcein staining but it is another experiment you need to do, to show percentage of in vitro maturation. Based on this sentence "They want aceto-orcein staining for sample of COCs after IVM", surely they know that oocyte will die after staining.
That's my idea
Dear Nguyen,
I agree with your opinion. However, it is difficult to carry out extra experiment or test the success of IVM with aceto-orcein staining for every study. However, it is important to determine the success of IVF system. Nevertheless, as you know, the processes to determine nuclear maturation stage of COCs following IVM can damage matured oocyte and detrimentally affect success of IVF. I want to know exact relationship between cumulus expansion and nuclear maturation of COCs. An other words, if we had 100 COCs with cumulus expansion after IVM, how much would COCs from hundred have reached to nuclear maturation ? I want to know the percentage of nuclear maturation from COCs with cumulus expansion.
Dear Umut,
I am assuming your question is related to bovine spp
Although the cumulus expansion and nuclear maturation may seem correlated at the end of the maturation time, in vitro this is more subjective in my opinion since the COCs at the periphery seem to be fully expanded as opposed to the ones remaining in the center of a particular group culture (they end up being mature too). We have done an extensive work on maturation of bovine oocyte and parthenogenetic activation. In our system, maturation rates measured by 1st polar body extrusion before ionomycine treatment were at least 90%. Please refer to the below article experiment one to check for the maturation rates.
The effects of conjugated linoleic acid isomers cis-9,trans-11 and trans-10,cis-12 on in vitro bovine embryo production and cryopreservation
I agree with Islam, using Hoechst will tell you the meiosis status of a denuded oocyte at a given time during IVM. However, I am afraid you would have to do this as a separate experiment. Your other option would be to perform ICSI if you are working with a different specie, once first polar body is determined.
Best wishes,
I have used aceto-orcein staining for my experiments in prepubertal sheep. Maturation is not really related to nuclear maturation. for example you can mature your temple oocytes in only TCM199 without expansion but you will have maturation. you can use 50 oocytes and use 10 of them for your staining and leave the rest for IVF.
Hi Vahid,
Thank you for your answer, you found that nuclear maturation may happen without cumulus expansion. However, some experts or researchers point out that expansion is not sing of nuclear maturation. So they want to carry out aceto-orcein staining in COCs with cumulus expansion in order to determine the success of IVM. Of course sampling is a good method to carry on IVF in the rest of COCs...
After more than 20 years daily IVF abd ICSI practice in human I can confirm that there is a ppor relation between cumulus expansion and meiotic resumption. In the early ages when there was no ICSI we tend to use cumulus expansion as a sign of oocyte maturity and quality we have experienced this is not very exact .
Hi,
I agree with the consensus of the comments above:
- cumulus expansion and meiotic maturation normally go hand in hand. But,
- cumulus expansion is a very poor measure of meiotic maturation, especially in an IVM context
- cumulus expansion and meiotic maturation can easily be dissociated, meaning you can have one without the other.
For example, GV-stage COCs put in simple culture medium will generally spontaneously meiotically mature without any cumulus expansion. The extent to which this is successful is very species dependent: about 80-90% of mouse, cow, sheep COCs will mature without cumulus expansion, whereas only 20-50% of human and pig COCs will mature without stimulation. FSH or EGF or EGF-like peptides will induce cumulus expansion and meiosis in these COCs. A further point is that, in an IVM context, cumulus expasnion does not equate with, and is not needed for, oocyte developmental competence. In fact under certain scenarios (eg IVM with high FSH), cumulus expansion is deleterious to further development.
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