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Effects of cyclic adenosine monophosphate modulators on maturation and quality of vitrified-warmed germinal vesicle stage mouse oocytes.

ABSTRACT: BACKGROUND:It is still one of the unresolved issues if germinal vesicle stage (GV) oocytes can be successfully cryopreserved for fertility preservation and matured in vitro without damage after warming. Several studies have reported that the addition of cyclic adenosine monophosphate (cAMP) modulators to in vitro maturation (IVM) media improved the developmental potency of mature oocytes though vitrification itself provokes cAMP depletion. We evaluated whether the addition of cAMP modulators after GV oocytes retrieval before vitrification enhances maturation and developmental capability after warming of GV oocytes. METHODS:Retrieved GV oocytes of mice were divided into cumulus-oocyte complexes (COCs) and denuded oocytes (DOs). Then, GV oocytes were cultured with or without dibutyryl-cAMP (dbcAMP, cAMP analog) and 3-isobutyl-l-methylxanthine (phosphodiesterase inhibitor) during the pre-vitrification period for 30 min. RESULTS:One hour after warming, the ratio of oocytes that stayed in the intact GV stage was significantly higher in groups treated with cAMP modulators. After 18 h of IVM, the percentage of maturation was significantly higher in the COC group treated with dbcAMP. The expression of F-actin, which is involved in meiotic spindle migration and chromosomal translocation, is likewise increased in this group. However, there was no difference in chromosome and spindle organization integrity or developmental competence between the MII oocytes of all groups. CONCLUSIONS:Increasing the intracellular cAMP level before vitrification of the GV oocytes maintained the cell cycle arrest, and this process may facilitate oocyte maturation after IVM by preventing cryodamage and synchronizing maturation between nuclear and cytoplasmic components. The role of cumulus cells seems to be essential for this mechanism.

SUBMITTER: Lee D

PROVIDER: S-EPMC6971999 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundWe had previously demonstrated that vitrification reduces the levels of certain phospholipid classes, and that oocytes from aged mice show a similar lipidome alteration, even without vitrification. In the current investigation, we examined if vitrification-warming of mouse oocytes from young and aged mice causes any changes in molecular aspects of lipid-associated features.MethodsMetaphase II (MII) stage oocytes were harvested from young (10-14-week-old) and aged (45-54-week-old) mice by a superovulation regime with PMSG followed by hCG. We examined the status of the intracellular lipid pool and the integrity of the plasma membrane by staining oocytes with BODIPY 500/510 and CellMask live dyes. Expression of lipid uptake- and necroptosis-associated genes was assessed by quantitative PCR analyses, in oocytes from young and old mice, before and after vitrification. Localization patterns of two crucial necroptosis proteins, phosphorylated MLKL (pMLKL) and phosphorylated RIPK1 (pRIPK1) were examined in mouse oocytes by immunofluorescence staining. Necrostain-1 (Nec1), an inhibitor of RIPK1, was used to examine if RIPK1 activity is required to maintain oocyte quality during vitrification.ResultsWe confirmed that vitrified-warmed oocytes from aged mice showed noticeable decrease in both CellMask and BODIPY 500/510 dyes. Among the lipid uptake-associated genes, Cd36 expression was higher in oocytes from aged mice. Necroptosis is a type of programmed cell death that involves damage to the plasma membrane, eventually resulting in cell rupture. The expression of necroptosis-associated genes did not significantly differ among groups. We observed that localization patterns of pMLKL and pRIPK1 were unique in mouse oocytes, showing association with microtubule organizing centers (MTOCs) and spindle poles. pMLKL was also localized on kinetochores of MII chromosomes. Oocytes treated with Nec1 during vitrification showed a decreased survival rate, indicating the importance of RIPK1 activity in oocyte vitrification.ConclusionsWe report that oocytes from aged mice show differential expression of CD36, which suggests that CD36-mediated lipid uptake may be influenced by age. We also show for the first time that pMLKL and pRIPK1 exhibit unique localization pattern in mouse oocytes and this may suggest role(s) for these factors in non-necroptosis-associated cellular processes.

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Effects of cyclic adenosine monophosphate modulators on maturation and quality of vitrified-warmed germinal vesicle stage mouse oocytes.

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