This study examined the concentration of cell-free mitochondrial DNA (cf-mtDNA) in porcine follicular fluid (FF) and explored whether the cfDNA level in the culture medium could reflect
mitochondrial dysfunction in cumulus cell-oocyte complexes (COCs). cfDNA concentration was higher in the fluid of small-sized follicles, compared to that in larger follicles. The length of
cfDNA ranged from short (152 bp) to long (1,914 bp) mtDNA in FF, detected by polymerase chain reaction (PCR). cfDNA concentration in FF significantly correlated with the mtDNA copy number in
FF but not with the number of one-copy gene (nuclear DNA) in FF. When the COCs were treated with the mitochondrial uncoupler, namely carbonyl cyanide m-chlorophenyl hydrazone (CCCP), for 2 h
and incubated for 42 h, subsequent real-time PCR detected significantly higher amount of cf-mtDNA, compared to nuclear cfDNA, in the spent culture medium. The mtDNA number and viability of
cumulus cells and oocytes remained unchanged. When the oocytes were denuded from the cumulus cells following CCCP treatment, PCR detected very low levels of cfDNA in the spent culture medium
of the denuded oocytes. In contrast, CCCP treatment of granulosa cells significantly increased the amount of cf-mtDNA in the spent culture medium, without any effect on other markers,
including survival rate, apoptosis of cumulus cells, and lactate dehydrogenase levels. Thus, cf-mtDNA was present in FF in a wide range of length, and mitochondrial dysfunction in COCs
increased the active secretion of cf-mtDNA in the cultural milieu.
Mitochondrial quality control is important for maintaining cellular and oocyte viability. In addition, aging affects mitochondrial quality in many cell types. In the present study, we examined how aging affects oocyte mitochondrial biogenesis and degeneration in response to induced mitochondrial dysfunction. Cumulus oocyte complexes were harvested from the ovaries of young (21-45 months) and aged (!120 months) cows and treated for 2 hours with 10 μM carbonyl cyanide-m-chlorophenylhydrazone (CCCP), or a vehicle control, after which cumulus oocyte complexes were subjected to in vitro fertilization and culture. CCCP treatment reduced ATP content and increased reactive oxygen species (ROS) levels in the oocytes of both young and aged cows. When CCCP-treated cumulus oocyte complexes were subsequently cultured for 19 hours and/or subjected to fertilization, high ROS levels in oocytes and a low rate of blastocyst development was observed in oocytes derived from aged cows. In addition, we observed differential responses in mitochondrial biogenesis to CCCP treatment between young and aged cows. CCCP treatment enhanced mitochondrial biogenesis concomitant with upregulation of SIRT1 expression in oocytes of young, but not aged, cows. In conclusion, aging affects mitochondrial quality control and recuperation of oocytes following CCCP-induced mitochondrial dysfunction.
GC number may be a useful marker for "good" follicles and oocytes, and the characteristics of the FFs determine granulosa cell number and oocyte developmental ability.
PurposeThe effect of supplementing maturation medium with follicular fluid (FF) was examined according to its non‐esterified fatty acid (NEFA) content or with a fatty acid mixture (FA‐Mix) on the developmental competence of oocytes, as well as the mitochondrial quality and quantity in the oocytes and cumulus cells.MethodPorcine oocytes from a slaughterhouse were used.ResultsThe FF or FA‐Mix in maturation medium increased the lipid content in both the oocytes and the cumulus cells, but the adenosine triphosphate content was differentially affected. The FF supplementation increased the mitochondrial DNA copy number, survival of cumulus cells, and rate of oocyte development to the blastocyst stage, whereas the FA‐Mix supplementation did not show these effects. The expression levels of GPC4,PFKP,PRDX3, and TFAM in the cumulus cells increased after FF supplementation, but the expression of GJA1 decreased, compared with the cells that were cultured without FF.ConclusionAdding FF and FA‐Mix to the maturation medium increased the lipid content in the oocytes and cumulus cells. The effects of FF on the cumulus cells and oocytes were not observed after FA‐Mix supplementation, indicating that the concentration of the NEFAs in the FF are closely associated with an ability to support oocyte maturation and the metabolism of cumulus cells and oocytes.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.