Effect of aging on mitochondria and metabolism of bovine granulosa cells.

This study investigated the effect of aging on mitochondria in granulosa cells (GCs) collected from the antral follicles of young and aged cows (25-50 months and over 140 months in age, respectively). When GCs were cultured under 20% O2 for 4 days, mitochondrial DNA copy number (Mt-number), determined by real-time PCR, increased throughout the culture period, and the extent of increase was greater in the GCs of young cows than in those of old cows. In a second experiment, GCs were cultured under 20% O2 for 24 h. Protein levels of TOMM20 and TFAM in GCs were lower in aged cows than in young cows, and the amount of reactive oxygen species and the mitochondrial membrane potential were higher, whereas ATP content and proliferation activity were lower, respectively. Glucose consumption and lactate production were higher in the GCs of aged cows than in those of young cows. When GCs were cultured under 5% or 20% O2 for 24 h, low O2 decreased ATP content and increased glucose consumption in GCs of both age groups compared with high O2; however, low O2 decreased the Mt-number only in the GCs of young cows. In conclusion, we show that aging affects mitochondrial quantity, function, and response to differential O2 tensions in GCs.

Effect of cryopreservation on the ability of granulosa cells to support in vitro development of oocytes derived from porcine early antral follicles.

Granulosa cells (GCs) contribute to oocyte development. The present study addressed the effect of cryopreservation on the ability of GCs to support oocyte development. GCs were collected from antral follicles. Oocyte granulosa cell complexes (OGCs) derived from early antral follicles were cultured with additional fresh-GCs or frozen-thawed-GCs for 14 days, and the developmental ability and characteristics of the oocytes grown in vitro were examined. Furthermore, fresh- or frozen-thawed-GCs were cultured for two days, and the effects of cryopreservation on the characteristics of GCs were examined. The developmental ability of blastocysts and the acetylation levels of H4K12 in oocytes grown in vitro did not significantly differ among the three culture conditions: OGCs cultured with additional fresh-GCs, frozen-thawed-GCs, or without additional GCs. Although both fresh- and frozen-thawed-GCs exhibited increased ATP content compared with that in oocytes developed without additional GCs, only fresh-GCs showed significantly increased lipid content in oocytes grown in vitro. ATP content, reactive oxygen content, mitochondrial membrane potential, and mitochondrial DNA copy number were greater in cultured frozen-thawed-GCs compared with fresh-GCs. In contrast, lipid content of cultured frozen-thawed-GCs was lower than that of fresh-GCs. Both fresh- and frozen-GCs support oocyte growth, but cryopreservation changes the properties of GCs in a manner that affects the energy status of oocytes grown in vitro.

Mitochondrial cell-free DNA secreted from porcine granulosa cells.

Several studies have proposed that cell-free DNA (cfDNA) is a potential biomarker present in follicular fluid (FF) for oocyte quality. Recently we reported that mitochondria-derived cfDNA (mt-cfDNA) closely reflects the amount of cfDNA in FFs. The present study investigated the mechanism regulating mt-cfDNA secretion from porcine granulosa cells. Oocytes and cumulus cell complexes or granulosa cells (GCs) were cultured in maturation medium for 24 or 48 h respectively. Then, nuclear-derived cell-free DNA (n-cfDNA) or mt-cfDNA contents in the spent medium were examined using real-time polymerase chain reaction. When 10 µM of MG132, a proteasome inhibitor, was added to the culture medium, cellular viability of both COCs and GCs decreased and n-cfDNA significantly increased in the culture medium, whereas mt-cfDNA significantly decreased. Supplementation of the culture medium with GW4869, an inhibitor of intracellular vesicle formation, significantly decreased the mt-cfDNA, whereas no effect was observed on n-cfDNA in the medium of both COCs and GCs. Furthermore, the addition of bafilomycin, an inhibitor of autophagy to the culture medium significantly increased mt-cfDNA in the culture medium. After filtration (0.22 µm) and centrifugation (23,000 g), the mt-cfDNA content of the medium decreased significantly. In conclusion, the proteasomal mitochondrial quality control system is upstream of mt-cfDNA secretion and autophagy plays a role in cellular digestion of mitochondrial DNA in the cytoplasm. It is further suggested that dsDNA is enclosed in certain vesicles or associated with small molecules and secreted into the medium.

Effects of resveratrol treatment on mitochondria and subsequent embryonic development of bovine blastocysts cryopreserved by slow freezing.

This study evaluated the effects of cryopreservation by slow freezing on the mitochondrial function, DNA integrity, and developmental ability of bovine embryos and examined whether resveratrol treatment of the frozen-thawed blastocysts improved embryonic viability. In vitro produced bovine embryos were subjected to slow freezing. After thawing, the ATP content and mitochondrial DNA integrity (mtDNA), determined by real-time PCR targeting short and long mitochondrial sequences, was found to be lower in frozen-thawed embryos than in fresh embryos, and mtDNA copy number was significantly reduced during the 24-hr incubation post warming. Furthermore, immunostaining against double-strand DNA revealed DNA damage in frozen-thawed embryos. When frozen-thawed embryos were incubated in the medium containing 0.5 µM resveratrol, SIRT1 expression, and survival rate of the embryos significantly improved compared with the vehicle-treated embryos. In addition, cell-free mtDNA content in medium was higher in case of resveratrol-treated embryos than of vehicle-treated embryos. In conclusion, slow freezing affects mitochondrial integrity and function in the blastocysts. In the frozen-thawed embryos, mitochondria were removed during post-thawing incubation and resveratrol enhanced the process, resulting in improved survivability of the embryos.

Follicular factors determining granulosa cell number and developmental competence of porcine oocytes.

PURPOSE: Granulosa cell (GC) number in follicles is a simple characteristic of follicles. The present study examined the hypothesis that follicular fluid (FF) determines GC number and oocyte developmental ability and revealed the molecular background determining the number of follicular GCs. METHODS: FF was collected from antral follicles (3-5 mm in diameter), after which the number of GCs per follicle was determined for each donor gilt using real time PCR targeting single copy gene. GCs were analyzed by next-generation RNA sequencing and IPA pathway analysis. RESULTS: When oocyte cumulus cell-oocyte-complexes (COCs) were cultured in maturation medium containing 10% of each individual FF, the rate of development to the blastocyst stage was significantly correlated with the number of GCs in the donor gilt. In addition, when GCs were cultured in medium containing FF, the proliferative activity of the GCs was also significantly correlated to the number of GCs in the donor gilt. Moreover, when the FFs were categorized based on the number of GCs in the follicle, it was found that supplementation of culture media with GC-rich FF improved the developmental ability of oocytes compared to those supplemented with GC-poor FF. RNA sequencing and a pathway analysis of GCs from GC-rich and -poor follicles revealed the key regulatory pathway determining GC number in follicles. CONCLUSION: 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.

Mitochondrial dysfunction in cumulus-oocyte complexes increases cell-free mitochondrial DNA.

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.

Non-esterified fatty acid-associated ability of follicular fluid to support porcine oocyte maturation and development.

PURPOSE: The 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. METHOD: Porcine oocytes from a slaughterhouse were used. RESULTS: The 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. CONCLUSION: Adding 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.

Maternal aging affects oocyte resilience to carbonyl cyanide-m-chlorophenylhydrazone -induced mitochondrial dysfunction in cows.

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.

Differential effects of mitochondrial inhibitors on porcine granulosa cells and oocytes.

Oocytes and granulosa cells rely primarily on mitochondrial respiration and glycolysis for energy production, respectively. The present study examined the effect of mitochondrial inhibitors on the ATP contents of oocytes and granulosa cells. Cumulus cell-oocyte complexes (COCs) and granulosa cells (GCs) were collected from the antral follicles of porcine ovaries. Treatment of denuded oocytes with either carbonyl cyanide m-chlorophenyl hydrazine (CCCP), antimycin, or oligomycin significantly reduced ATP content to very low levels (CCCP, 0.12 pM; antimycin, 0.07 pM; and oligomycin, 0.25 pM; P < 0.05), whereas treatment with a glycolysis inhibitor (bromopyruvic acid, BA) had no effect. Conversely, the ATP content of granulosa cells was significantly reduced by treatment with the glycolysis inhibitor but was not affected by the mitochondrial inhibitors (ATP/10,000 cells; control, 1.78 pM and BA, 0.32 pM; P < 0.05). Reactive oxygen species (ROS) generation after CCCP treatment was greater in oocytes (1.6-fold) than that seen in granulosa cells (1.08-fold). Oocytes surrounded by granulosa cells had higher ATP levels than denuded oocytes. Treatment of COCs with CCCP reduced, but did not completely abolish, ATP content in oocytes (control, 3.15 pM and CCCP, 0.52 pM; P < 0.05), whereas treatment with CCCP plus a gap junction inhibitor, 18α-glycyrrhetinic acid, and CCCP decreased the ATP content to even lower levels (0.29 pM; P < 0.05). These results suggest that granulosa cells are dependent on glycolysis and provide energy to oocytes through gap junctions, even after treatment with CCCP.