The use of resveratrol decreases liquid-extend boar semen fertility, even in concentrations that do not alter semen quality.

In vitro and in vivo assays were conducted to investigate the effects of trans-resveratrol (RVT) on liquid-extended boar semen during 72 h of storage at 17 °C. Thirty-six ejaculates were collected from six boars, evaluated, and extended. RVT was then added at the indicated treatment concentration (0, 0.01, 0.1 or 1 mM), and the ejaculates were cooled to 17 °C and evaluated at 0, 24, 48, and 72 h. Samples were evaluated for sperm motility, kinetics, plasma and acrosome integrity, mitochondrial membrane potential, anion superoxide levels, lipoperoxidation, and antioxidant enzyme activity. In the follow-up experiment, twenty-eight gilts were fixed-time inseminated with 0 or 0.01 mM RVT liquid-extended boar semen. After five days, they were slaughtered, and their reproductive tracts were recovered. The embryos were collected, and the pregnancy, fertility, and viable embryo rates were calculated. In the in vitro assays, total motility, plasma and acrosome membrane integrity, mitochondrial membrane potential, anion superoxide levels, and lipoperoxidation did not change at any of the evaluation times with the use of RVT up to 0.01 mM. RVT decreased SOD activity without changes in GPx. RVT used at 1 mM showed harmful effects for almost all evaluated parameters. For the in vivo assay, the same pregnancy and fertility rates were observed for both groups, while the viable embryo rate was three-fold lower in the 0.01 mM group than in the 0 mM group. The results showed a dichotomous effect of RVT; a low concentration was not harmful in vitro but was catastrophic for embryo viability.

Evidence of Selection Against Damaged Mitochondria During Early Embryogenesis in the Mouse.

There is evidence of a purifying filter acting in the female germline to prevent the expansion of deleterious mutations in the mitochondrial DNA (mtDNA). Given our poor understanding of this filter, here we investigate the competence of the mouse embryo to eliminate dysfunctional mitochondria. Toward that, mitochondria were damaged by photoirradiation of NZB/BINJ zygotes loaded with chloromethyl-X-rosamine (CMXRos). The resultant cytoplasm was then injected into C57BL/6J zygotes to track the levels of NZB/BINJ mtDNA during the preimplantation development. About 30% of NZB/BINJ mtDNA was present after injection, regardless of using photoirradiated or non-photoirradiated cytoplasmic donors. Moreover, injection of photoirradiated-derived cytoplasm did not impact development into blastocysts. However, lower levels of NZB/BINJ mtDNA were present in blastocysts when comparing injection of photoirradiated (24.7% ± 1.43) versus non-photoirradiated (31.4% ± 1.43) cytoplasm. Given that total mtDNA content remained stable between stages (zygotes vs. blastocysts) and treatments (photoirradiated vs. non-photoirradiated), these results indicate that the photoirradiated-derived mtDNA was replaced by recipient mtDNA in blastocysts. Unexpectedly, treatment with rapamycin prevented the drop in NZB/BINJ mtDNA levels associated with injection of photoirradiated cytoplasm. Additionally, analysis of mitochondria-autophagosome colocalization provided no evidence that photoirradiated mitochondria were eliminated by autophagy. In conclusion, our findings give evidence that the mouse embryo is competent to mitigate the levels of damaged mitochondria, which might have implications to the transmission of mtDNA-encoded disease.

Maternal transmission of mitochondrial diseases.

Given the major role of the mitochondrion in cellular homeostasis, dysfunctions of this organelle may lead to several common diseases in humans. Among these, maternal diseases linked to mitochondrial DNA (mtDNA) mutations are of special interest due to the unclear pattern of mitochondrial inheritance. Multiple copies of mtDNA are present in a cell, each encoding for 37 genes essential for mitochondrial function. In cases of mtDNA mutations, mitochondrial malfunctioning relies on mutation load, as mutant and wild-type molecules may co-exist within the cell. Since the mutation load associated with disease manifestation varies for different mutations and tissues, it is hard to predict the progeny phenotype based on mutation load in the progenitor. In addition, poorly understood mechanisms act in the female germline to prevent the accumulation of deleterious mtDNA in the following generations. In this review, we outline basic aspects of mitochondrial inheritance in mammals and how they may lead to maternally-inherited diseases. Furthermore, we discuss potential therapeutic strategies for these diseases, which may be used in the future to prevent their transmission.

Effects of melatonin during IVM in defined medium on oocyte meiosis, oxidative stress, and subsequent embryo development.

Melatonin may have beneficial effects when used in oocyte maturation and embryo development culture. The effect of melatonin during IVM on meiosis resumption and progression in bovine oocytes and on expression of antioxidant enzymes, nuclear fragmentation and free radicals, as well as on embryo development were assessed. Cumulus-oocyte complexes were matured in vitro with melatonin (10(-9) and 10(-6) M), FSH (positive control), or without hormones (negative control) in defined medium. Maturation rates were evaluated at 6, 12, 18, and 24 hours. Transcripts for antioxidant enzymes (CuZnSOD, MnSOD, and glutathione peroxidase 4 (GPX4)) in oocytes and cumulus cells, nuclear fragmentation in cumulus cells (TUNEL) and reactive oxygen species levels in oocytes (carboxy-H2 difluorofluorescein diacetate) were determined at 24 hours IVM. Effect of treatments on embryo development was determined after in vitro fertilization and culture. At 12 hours, meiosis resumption rates in FSH and melatonin-treated groups were similar (69.6%-81.8%, P > 0.05). At 24 hours, most oocytes were in metaphase II, with FSH showing highest rates (90.0%, P < 0.05) compared with the other groups (51.6%-69.1%, P > 0.05). In cumulus cells, MnSOD expression was higher in FSH group (P < 0.05) whereas Cu,ZnSOD transcripts were more abundant in melatonin group (10(-6)M; P < 0.05). Nuclear fragmentation in cumulus cells was highest in controls (37.4%/10,000 cells; P < 0.05) and lower in FSH and 10(-6)M melatonin (29.4% and 25.6%/10,000 cells, respectively). Reactive oxygen species levels were lower in oocytes matured with 10(-6)M melatonin than in control and FSH groups (P < 0.05). Embryo development from oocytes matured only with melatonin was similar to those matured in complete medium (P > 0.05). In conclusion, although melatonin during IVM in a defined medium does not stimulate nuclear maturation progression it does stimulate meiosis resumption and such treated oocytes support subsequent embryo development. Melatonin also shows cytoprotective effects on cumulus-oocyte complexes.