Oocytes - Science topic
Female germ cells derived from OOGONIA and termed OOCYTES when they enter MEIOSIS. The primary oocytes begin meiosis but are arrested at the diplotene state until OVULATION at PUBERTY to give rise to haploid secondary oocytes or ova (OVUM).
Questions related to Oocytes
Hello everyone. I would like to discuss my ICSI procedure. I´ve been learning ICSI method for a year within my thesis, that focused on interspecific ICSI (specifically mouse oocytes and goat sperm). My problem is, that i can not reach satisfying numbers of viable injected oocytes. A lot of oocytes break and flow out during an injection. If they survive, some of them die later during cultivation in incubator. I hope you can understand, that I´m still a rookie, but I hope you can give me some good advices. The procedure is as follows:
- PMSG stimulation
- hCG stimulation (48 h after PMSG)
- Isolation of oocytes (16-17h after hCG)
- --- for isolation: M2 medium (10ml M2-HEPES + 10 mg BSA, stored in 4°C, filtered, heated to 36,8°C 1/2 h before use)
- Removal of cumulus cells - in drops of hyaluronidase (2 mg HYA + 2 ml PBS, heated before use)
- --- I have always been careful about this step for limited time (few seconds) that oocytes spend in drops. If cumulus cells have been removed, oocytes are washed in fresh drops of same M2 medium. Then oocytes are moved to drops of culture medium - usually MEM (40 mg BSA + 10 ml MEM + Na pyruvate gentamicin 100 μg).
- Oocyte "rest" - in 4well in MEM medium in incubator with 5% CO2 . Recovering from isolation for 1/2 h.
- ICSI - oocytes are moved from MEM to M2 medium. First in fresh drops outside, then M2 drops in manipulation dish (covered with mineral oil).
- Sperm that is used for this procedure is frozen, so it is thawed and moved to M2 medium (centrifuged and washed from cyroprotectants)
- Piezo system is used for this procedure. Injection pipette contains minimal amount of mercury. Pulses regulation depending on oocytes condition. Trying to use the bigger pulse for breaking through ZP and smaller pulse to get into cytoplasm. Than pull injection pipette slowly from oocyte.
Could you please tell me, if there is any critical thing in this procedure that could cause this problem. My master uses same protocol (except that he is a pro with many years of experience). I really understand that it is most likely my "hands" that make mistakes. So this is a little bit challenge for me, but I believe that you can understand that everyone had to start and learn.
Thank you all for your answers :)
I am interested in creating plasma membrane blebs from Xenopus oocyte by hypertonic treatment. Does any one have experience in doing so? The most important question would be to as do formed blebs harbour membrane expressed proteins?
Any help is greatly appreciated!
Thanks and kind regards
I've been cryo-sectioning Xenopus oocytes and find these large internal branching structures in all of them (see attached image). I haven't been able to find a reference to it in the literature. I was tempted to say that it was ER, but I don't think it is. Does anyone have information on what this structure is?
I'm using JC-1 to measure the mitochondrial membrane potential by laser confocal microscopy. I used 100uM of FCCP as a positive control. Surprisingly, it caused lower green to red ratio and was significantly lower than the untreated cells. My cells are cryopreserved mice oocyte. On the other hand, FCCP did work and caused an elevation in calcium using Fluo 3 AM and caused plasmalemmal membranes depolarization.
As we know that the sperms function test is widely used for the management of male factor issues worldwide. However, similar parallel test is not available for the management of egg factor issue in female. Do the number of oocytes available for the purpose or the unavailability of the potential biomarkers for oocyte quality and other factors limit the development of oocyte function test? Kindly give your expert opinion.
Hello, can someone help me? I am just random undergrad interested in oocytes and early embryos but I know almost nothing about their in vitro cultivation. What is the current state of the art of this topics? Where can I find what are the ingredients and what do they affect? Could you recommended me some reviews, articles, brochures, websites, anything about this topics?
Thank you very much!
Well, we read a lot about how in vitro manipulation of oocytes can induce stress and therefore reduce their potential or as people call it "quality". I've been looking into the studies mostly, they focus on the oxidative stress and reactive oxygen species relation with calcium regulation. Other studies investigated the heating as a stress factor. Well, how they established that this oocyte is healthy and this one is stressed? On what basis? What kind of methods they used? Are the polscope data useful?
Our long standing understanding for mammalian ovary is that it has fixed number of germ cells and duty of the ovary is to ovulate high quality ovum for successful fertilization and early embryonic development. However, growing studies suggest that the oocytes can be generated from Oocyte like stem cell/neo-oogenesis/adult oogenesis and fertilized in vitro. Therefore, it is important tom know whether oocytes generated from these sources are as good as the follicular oocytes collected from mammals under natural conditions. Kindly give your opinion on the genomics, proteomics and metabolomics aspects of ocytes generated from Oocyte like stem cell/neo-oogenesis/adult oogenesis.
Hello - I need to retrieve GV stage oocytes from mice ovaries. I do not know where to get the small tool that is used to mush the ovaries into small pieces. I do not know how to fabricate it either. Does anyone know if it is commercially available? If not, do you have any spares you are willing to part with? Thank you very much in advance.
Recent evidences suggest that mitochondria play important roles in determining the quality and fate of the oocytes in several mammalian species. Changes in shape, number as well as the distribution pattern of mitochondria alter oocyte physiology. More scientific discussion and suggestions are needed in order to develop a new biomarker for for the improvement of assisted reproductive technology outcome.
The maturation promoting factor (MPF) is a key regulator of cell cycle that has regulatory subunit (Cyclins) and catalytic subunit (Cdks). Dissociation and degradation of specific cyclin destabilizes MPF, while its association with specific Cdk activate MPF.
I want to culture oocytes in vitro and analyze gene expression from primordial follicles and primary follicles in mice, but am unsure how to collect isolated oocytes and granulosa cells. So far I've only seen articles with protocols involving hyaluronidase and mechanical methods for denuding the oocyte. I want to isolate the oocytes from granulosa cells in mice. Does anyone have any particular methods that they could recommend to me please
I am doing a mammalian oocytes maturation, followed by nuclear meiotic evaluation using confocal microscopy, I mount the oocyte over 90% glycerol in a double sided adhesive tape, and I am curious because I don't want to smash the oocytes under the cover slip. is there any recommendation for a good commercial wax cushion to be used in this case. (oocyte diameter is 100 micron).
I would like to ask you what is the best model to mimic human corpus luteum functions in vitro. I saw in literature that Granulosa Cells can be isolated from follicular liquid during oocyte withdraw for IVF. What would be the best protocol to differentiate these cells in granulosa-lutein cells? would it be reliable and scientifically accepted?
I recently started my masters thesis and for that matter I will patch oocytes. I need to prepare some solutions for the procedure and I need to get Cs-glutamate. I cannot find anything on the internet about how to make it. It is also not possible to order it anywhere.
Does anyone know how to make or get Cs-Glutamate?
Xenopus laevis oocytes are used in electrophysiology to make transient expression of injected mRNA fragments. Through the technique of 2-electrodes voltage-clamp we can record the physiological phenomena that originate from the activity of the generated protein that has been embedded in the oocyte membrane. Depending on its activity we will observe hyperpolarization or repolarization of the membrane, in the presence of different external stimuli (different concentrations of anions, amino acids, organic acids, etc.).
In our case, electrophysiology recordings in Xenopus laevis were assayed. A plant's membrane transporter working in an animal cell membrane.
But, in young and old, well-fed, and apparently healthy frogs, sometimes the experiments could not be as brilliant as on other occasions. The oocytes were of poor quality, without showing that polarized two-color differentiation, and after injecting the RNA they exploded most of them. Surviving oocytes showed strange electrical behaviors, mostly related to passive endogenous chloride input channels and ended up lysing soon.
Have you had any similar experiences? If you have an explanation I would be very grateful. We are trying to "dust off" old jobs that were abandoned in a drawer.
I want to thank Dr. Omar Homero Pantoja from IBT-UNAM (Cuernavaca, Mexico) for teaching me during my stay in his electrophysiology laboratory.
#xenopus #oocytes #2EVC #electrophysiology #recordings
Hi guys! I would like to know if someone could help me to solve a problem the following problem: After the parthenogenetic activation of oocytes, zygotes are degenerating after 72h of activation. I made some changes in the SOF environment, but there was no positive result. Who can help me, I appreciate it!!
Must all groups in chi-square have the same number of for example cells (or embryos etc.)? We analyzed 30, 28, 37 and 36 oocytes in experimental groups.
I would like to avoid transfering oocytes from one solution to another by micropipeting and I am wondering if I can attach them somehow on a slide and employ fixation and staining procedure on them for immunocytochemisty.
Thank you in advance.
I am looking forward for your suggestions if possible.
Hello everyone! I am relatively new to the zebrafish world, and I am hoping to ask some questions about their reproductive biology. After breeding my fish, I often find what appear to be "smushed" or hollow-looking "eggs". I am not sure what they are, but suspect they are perhaps some sort of underdeveloped oocyte or follicle-related tissue? I am pretty certain they are not scales as they have some thickness to them; the shapes/sizes are also inconsistent.
I've attached some photos. In the larger photo, I have circled the "smushed" egg (Red) as well as an unfertilized oocyte (Yellow) for comparison, next to fertilized oocytes (uncircled). There is a close up image also attached. If anyone can direct me to some resources to learn more that would be great as well.
There are several studies that have shown that different types of stem cells (mesenchymal stem cells, embryonic, endometrial, and ovarian stem cells) can be differentiated into both spermatozoa and oocytes in vitro, proving their potential clinical use in management and cure of infertility issues. Can these trials be applicable?
Open pulled straws which are made by manually pulling heated French mini straws usually get contaminated during processing and after that how they can be sterilized. I mean the method recommended without any deleterious effect on the quality of oocytes and embryos.
In this case-control study, 60 patients with in vitro fertilization (IVF)/intracytoplas- mic sperm injection (ICSI) indication were subdivided into 3 groups as follow: 20 subjects were assigned to control (fertile women with male infertility history) group, 20 subjects with PCOS were insulin resistant (IR) and 20 subjects with PCOS were insulin sensitive (IS). After puncture, retrieved oocytes were classified into metaphase II (MII) as mature and in metaphase I (MI) or germinal vesicle stage (GV) as immature. Regres- sion analyses were used to explore the association between MII oocyte number and demographic and clinical variables. LH/FSH ratio was significantly higher in PCOS-IR women compared to controls but not significantly dif- ferent from that of PCOS-IS group. PCOS-IR women had lower MII oocyte number compared with that of controls. According to multiple regression analysis, the number of previous assisted reproductive technology (ART) cycles was negatively associated with the number of MII oocy
Has anyone been able to successfully cryopreserve coral oocytes? I've only found one case for a Gorgonian (Junceella juncea) through vitrification (Tsai et al. 2015, attached below). I know that in some cases some researchers (personal communications) have been able to thaw and retrieve from liquid nitrogen, but though they are still 'alive', they loose their ability to be fertilized (infertile).
I appreciate your help providing me with your answers and/or experience in this matter.
I bought a stereo microscope with led lighting and it makes very difficult to see oocytes /embryos. What microscope may you suggest for this kind of research? Thanks.
I want to know that can we use the cryotop device for vitrification in oocytes once we have thawed the already vitrified oocytes using this device...Is it recommended or not.....
Not sure about breeding strategy,
To speed up offspring production with the required genotype (I only need hetero and hetero share the same phenotype with the WT), is it ok to set up breeding pairs:
homo mutant (male) cross with WT (female)
and homo mutant (female) cross with WT (male),
which can give me the desired genotype 100 % than hetero crossing at a Modelian ratio, let us forget the controls from one littermate for the time being that must be used for the experiment.
I guess my question is how to know whether there is individual variance in the offspring because of oocyte/sperm coming from different genotype or not.
I want to inject equine oocytes using the femtojet but in the manual there is no explanation for the volume of injection. Only for the pressure and time. Does anyone know how to use both to determine the volume?
During the process of oocyte maturation, asymmetric divisions result in segregation of the maternal genome and highly asymmetric partition of the cytoplasm, which generates the small polar body and the large oocyte. Why does the polar body stop cell division even after parthenogenetic activation?
Hi. I want to do patch clamp on mouse oocyte. I have used poly L-lysine and either matrigel but no attachment was observed. Anybody knows another solution?
We want to extract RNA from small number of oocytes for RNA-seq. I am afraid that I lose RNA with column extractio. I am thinking about using Tirzol. Any idea?
I would like to purchase an antibody that works for ICC on oocytes. However, I have found the antibodies for WB only. So if they are possible to use for ICC, I want to perform with them. Those who know well on this, please provide me any references or reasons that I can use them. Thank you! Also, if the antibodies for WB are monoclonal, can that be possible for ICC?
I am struggling to find a way to measure oocytes on histological sections with the nucleus while excluding those without the nucleus. Since the measures would involve the whole histological section (i.e. a few hundreds of oocytes to manually be measured) I am trying segmenting and/or thresholding the pictures on imageJ, but without success. The idea underlying this approach is to find a way to automatically detect the "objects" I am specifically interested in.
I've already tried ImageJ Trainable Weka Segmentation and MorphoLibJ.
Here a couple of pictures of ovaries DAPi-stained and what I'd roughly like to obtain. Red and yellow circles for oocytes with and without nucleus, respectively.
Thank you in advance.
I am working with embryos and oocytes, i am confused about how many an which internal controls to use for qPCR?
in literature, i have seen only one internal control has been used and study is published in reputed journal. but i am not getting clear idea.
I'm looking for an effective way to clean some COOK® Flexipet capillaries (100 um of inner diameter).
We tried by flushing ethanol and dH2O using a insulin syringe, with little results. Is it possible to clean them better (maybe with stronger mixes such as RCA1 or Piranha) given that they are made of polycarbonate?
We have been trying to collect metaphase II oocytes from mice for some time. We tried a variety of ways for super ovulation. In each experiment we wash oviducts but we can hardly find any oocytes. I need your advice. Thank you in advance
My questions are about meiosis chromosome spreads.
My experiment is about a gene which can result in primary ovarian insufficiency perhaps through meiotic prophase I abnormality.
I have read some papers and protocols about this experimental technique. I can replicate the protocol but cannot recognize the 5 substages of meiosis prophase I (so as the first figure below).
And I also have to identify the wildtype and mutant oocyte chromosome patterns ,however, I’ve no idea about the abnormality or normal chromosome pattern, which is as indicated below (the second figure below). So I want to get help from you about the specific instructions on the identification of substages of meiosis prophase I and the normal patterns of the chromosome. Literature, books and personal experience will be deeply appreciated.
Im in Australia and we haven’t been able to import Folligon in for a few months now and all the alternative don’t seem to work on our mice. Does anyone have any other drug suggestions?
We use C57.B6/CBA females at 3 weeks of age. We inject 48 hours apart with 5IU and collect 13-15 hours later to collect MII oocytes.
We havw tried pregnecol as an alternative but no luck.
We will be collecting ovaries and surrounding tissues from slaughtered animals for oocyte collection. One of the goals is also to measure the diameter of arteries to ovary by ultrasound in a water bath. Do we need to ligate the vessels and fill them with fluid in order to be able to see them? Does anyone have suggestions/ a protocol for this?
I am going to study the response of cumulus cells after using a kind of media. Considering the diffusion may associated with the volume of the cell group, I need to measure the volume of human cumulus cells collected from immature COCs, after removing the oocyte. The cumulus cells are still connected together. Does anyone have any ideas?
I have nucleolus samples isolated from oocytes. If someone has an experience with total RNA isolation from such samples for NGS, please share or advise.
Could the transfer of embryos (or oocytes and zygotes) between different culture media induce alterations in their cytoskeletal structure leading to cell division failure?
Thanks in advance.
We all struggle to define and reach the best criteria to define and standardize sperm morphology, these can be seen from variations in quality assessments worlwide. Surprisingly, we don't worry to much about the quality of morphoogy of TESE sperm cells.
I would like to have expert opinions about this issue.
Thanks to all of you for joining the discussion and providing insights.
In vitro maturation of human oocytes could be an attractive strategy for in vitro fertilization treatment. However, current in vitro maturation protocols unfortunately produce oocytes with a poor clinical outcome. Over the last 30 years, advances in culture conditions have continuously improved IVM's efficiency but even now in-vitro maturation does not support all nuclear/cytoplasmic changes that occur physiologically as a result of ovulatory stimulus in vivo. Adding EGF family molecules (e.g. amphiregulin, epiregulin) to the culture media increases the IVM rate of immature human oocytes. Additionally, brain-derived neurotrophic factor (BDNF) and glial-cell-derived neurotrophic factor (GDNF) also could improve the IVM outcome. Delaying the maturation process by adding cAMP or kinase inhibitors is another option to combine with cytoplasmic maturation. Do you have experience with these supplements? What other options do you apply in your practice?
Hi,I have recently started working on mitochondria patch clamp in xenopus oocytes. I am trying to find the best osmolarity for my solutions.However, since I cannot find the exact value for cytoplasmic osmolarity within the oocytes i am having a hard time.
Thank you for your help.
I want to classify compounds as inhibitors or non-inhibitors for some transport protein.
The experimental results were xx fmol/oocyte/min or fmol/oocyte/hr. I have never done such experiment and don't know what it means.
1. How can I classify them with the unit of fmol/oocyte.
Normally, if a compound has a IC50 < 100 microM or under 100 microM it has 50% inhibition, I'll classify as inhibitor.
And I don't know how to classify them according to their uptake . Whether less than 80% or 50%?
2. Is uptake means 1-inhibition? If not, how to classify according to the uptake.
Sometimes, the results are not absolute value. Maybe the inhibition is 50% of control.
3. Can I regard them as the same and check whether the inhibition is higher than 50% (of control)?
My undergrad thesis is all about contamination of water buffalo oocytes in the in vitro culture. Now, i've been researching for a possible antimycotic which I can use as a treatment. However, I've found Amphotericin B but it's too expensive, now I'm considering Ketoconazole as an alternative.
Does anyone tried using this? And may I know the concentration of it? Or could you suggest any other antimycotic which are not expensive?
This is my undergraduate thesis as it will tackle the contaminants in the in vitro culture of oocytes. Furthermore, my professors recommended me to use Molecular Biology to identify these fungal contaminants and prevent them when I have already identified them. As for the prevention, what is the best measure you can advice to me? Can I use fungicide or just stick with the sterility of the laboratory?
During in vitro maturation of "camel" cumulus-oocyte complexes, I frequently noticed that cumulus cells start to attach to IVF non-treated dishes within few hours of culture.
I have not observed this attachment during my IVM work in bovine and porcine oocytes.
I think this attachment affects the quality of oocytes.
Do you have any explanation for that?
I wanted to investigate about apoptosis in mouse oocytes, but it's a new field to me (both apoptosis and mouse oocytes- I only worked with human cell lines before), so I hesitated to choose the good options to check apoptosis in mouse oocytes. I saw people often use COMET assay, TUNEL assay, Anexin V (flow cytometry or immnufluorescence), Western blot with PARP or casp 3 cleavage in different studies. Are there any advantages or disadvantages of those to use in mouse oocytes? (From what I see Anexin V is used on non-fixed cells, while COMET/TUNEL can be on fixed cellsbutI'm not sure for oocytes if it's better to go with fixed or non-fixed methos. And Western blot and Flow cytometry might be hard since I need to have a high number of oocytes).
I am going to simulate mechanical aspects of patch-clamp process in Finite Element Software, but I do not know the approximate size of the micro-pipette and oocytes which were used in such studies. Information like approximate or typical diameter of the Xenopus oocyte and inner/outer diameter of pipette tip would help.
Attached is an example of papers I am dealing with
So recently I injected a batch of xenopus laevis oocytes with some cRNA for some transport experiments. I noticed that there were quite a bit of internal content coming out right after the injection, but nothing too abnormal. After 12 hours or so, I checked the oocytes again and noticed that around 80% of them continued having intracellular content leaking out of the injection site and they soon turned pale and became necrotic. I've been doing oocyte experiments using the same technique for years and this is the first time I encounter such situation. I am wondering what is going on. If you can contribute some thoughts, it will be greatly appreciated.
Which protocol is best for preparation of porcine oocyte and iPS extracts for treating donor cell before SCNT?
Hey there. I need to isolate stem cells from mouse fetal membrane, for a project I'm working on. but I know nearly nothing about it, only a little about mating a male and female mice and pregnancy duration! but exactly how and when can I extract fetal membrane from pregnant mice and could it be after breeding the babies? I hope it would.
waiting.thanks a lot.
I am looking for a protocol optimized for the DNA (RNA-free) isolation of mammalian (preferentially mouse) MII oocytes.
I have different pools of 20-80 oocytes and would like to proceed with DNA-specific analysis.
I performed some bibliographic review and I see every different paper with a different protocol, so I thought it would be useful to ear someone with experience in this particular protocol.