RESUMO

BACKGROUND: The evaluation of the DNA damage generated in cumulus cells after mature cumulus-oocyte complexes vitrification can be considered as an indicator of oocyte quality since these cells play important roles in oocyte developmental competence. Therefore, the aim of this study was to determine if matured cumulus-oocyte complexes exposure to cryoprotectants (CPAs) or vitrification affects oocytes and cumulus cells viability, but also if DNA damage is generated in cumulus cells, affecting fertilization and embryo development. RESULTS: The DNA damage in cumulus cells was measured using the alkaline comet assay and expressed as Comet Tail Length (CTL) and Olive Tail Moment (OTM). Results demonstrate that oocyte exposure to CPAs or vitrification reduced oocyte (75.5 ± 3.69%, Toxicity; 66.7 ± 4.57%, Vitrification) and cumulus cells viability (32.7 ± 5.85%, Toxicity; 7.7 ± 2.21%, Vitrification) compared to control (95.5 ± 4.04%, oocytes; 89 ± 4.24%, cumulus cells). Also, significantly higher DNA damage expressed as OTM was generated in the cumulus cells after exposure to CPAs and vitrification (39 ± 17.41, 33.6 ± 16.69, respectively) compared to control (7.4 ± 4.22). In addition, fertilization and embryo development rates also decreased after exposure to CPAs (35.3 ± 16.65%, 22.6 ± 3.05%, respectively) and vitrification (32.3 ± 9.29%, 20 ± 1%, respectively). It was also found that fertilization and embryo development rates in granulose-intact oocytes were significantly higher compared to denuded oocytes in the control groups. However, a decline in embryo development to the blastocyst stage was observed after CPAs exposure (1.66 ± 0.57%) or vitrification (2 ± 1%) compared to control (22.3 ± 2.51%). This could be attributed to the reduction in both cell types viability, and the generation of DNA damage in the cumulus cells. CONCLUSION: This study demonstrates that oocyte exposure to CPAs or vitrification reduced viability in oocytes and cumulus cells, and generated DNA damage in the cumulus cells, affecting fertilization and embryo development rates. These findings will allow to understand some of the mechanisms of oocyte damage after vitrification that compromise their developmental capacity, as well as the search for new vitrification strategies to increase fertilization and embryo development rates by preserving the integrity of the cumulus cells.

RESUMO

BACKGROUND: Stress has been considered as one of the causes of decreased reproductive function in women. However, direct evidence of the effect of chronic stress on oocytes depending on estrous cycle phases is limited. OBJECTIVE: The present study aimed to evaluate the impact of chronic stress on the viability, integrity, and maturation of rat oocytes depending on estrous cycle phases, specifically proestrus, estrus, and diestrus. METHODS: For this purpose, adult female rats were stressed daily by cold water immersion (15 °C) for 30 consecutive days. RESULTS: In chronically stressed female rats, irregular estrous cyclicity, increased corticosterone levels, decreased oocyte viability, and an increased percentage of abnormal oocytes were obtained in all the estrous cycle phases, resulting in reduced oocyte maturation during proestrus. CONCLUSION: Oocyte maturation disturbed by chronic stress is a crucial factor by which chronic stress disrupts female reproduction.

RESUMO

Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at -196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.

RESUMO

Parabens (PBs) are compounds widely used in industry for food and personal care products as antimicrobials and preservatives. Their indiscriminate use has resulted in their detection in different ecosystems so that humans and other organisms are highly exposed. Methylparaben (MePB), compared with other PBs, is mostly detected in food, personal care and baby care products. PBs could be linked to the generation of hormonal disorders and fertility impairment since their recent classification as endocrine disruptors. The knowledge of the effects that MePB can exert is of great importance as, in terms of reproduction, information is limited. Therefore, the objective of the present study was to evaluate the effect of MePB on porcine oocyte viability and in vitro maturation (IVM), as well as to determine the lethal concentration at 50% (LC50 ) and the maturation inhibition concentration at 50% (MIC50 ). Oocytes were exposed to different MePB concentrations 0 (control), 50, 100, 500, 750 and 1000 µm during 44 h of IVM. Cytoplasmic alterations and reduced cumulus cell expansion were observed in oocytes exposed to MePB; however, viability was not affected. In addition, oocyte maturation decreased in a concentration-dependent manner after exposure to MePB. The estimated LC50 was 2028.38 µm, whereas MIC50 was 780.31 µm. To our knowledge, this is the first study that demonstrates that MePB altered porcine oocyte morphology, and caused a reduction in cumulus cell expansion, both of which resulted in decreased oocyte maturation. Therefore, MePB exposure may be one of the factors involved in fertility impairment in mammals, including that of humans.

Assuntos

RESUMO

Perfluorooctanoic acid (PFOA) is a member of the perfluoroalkyl acid family of compounds. Due to the presence of strong carbon-fluorine bonds, it is practically nonbiodegradable and highly persistent in the environment. PFOA has been detected in the follicular fluid of women, and positively associated with reduced fecundability and infertility. However, there are no reports concerning the experimental evaluation of PFOA on oocyte toxicity in mammals. The aim of the present study was to determine if PFOA is able to induce oxidative stress in fetal ovaries and cause apoptosis in oocytes in vitro. In addition, since inhibition of the gap junction intercellular communication (GJIC) by PFOA has been demonstrated in liver cells in vivo and in vitro, the effect of PFOA on the GJIC between the oocyte and its supportive cumulus cells was studied. Results show that PFOA induced oocyte apoptosis and necrosis in vitro (medium lethal concentration, LC50 = 112.8 µM), as evaluated with Annexin-V-Alexa 508 in combination with BOBO-1 staining. Reactive oxygen species (ROS) levels, as assessed by DCFH-DA, increased significantly in fetal ovaries exposed to » LC50 (28.2 µM, a noncytotoxic and relevant occupational exposure concentration) and LC50 PFOA ex vivo. This perfluorinated compound also caused the blockage of GJIC in cumulus cells-oocyte complexes (COCs) obtained from female mice exposed in vivo, as evaluated by calcein transfer from cumulus cells to the oocyte. The ability of PFOA of disrupting the GJIC in COCs, generating ROS in the fetal ovary and causing apoptosis and necrosis in mammal's oocytes, might account for the reported association between increasing maternal plasma concentrations of PFOA with reduced fertility in women.

Assuntos

RESUMO

BACKGROUND: Most studies carried out to evaluate recovery and development after porcine oocyte vitrification, reported better rates when cryopreserved in embryonic development stages or zygotes, but not in immature oocytes. For this reason, many studies are performed to improve immature oocyte vitrification protocols testing the use of different cryoprotectant concentrations, cooling devices, incubation times; but only a few of them have evaluated which fertilization procedure enhances blastocyst rates in vitrified oocytes. Therefore, this study was aimed to evaluate: 1) if the sperm selection with hyaluronic acid (HA) or polyvinylpyrrolidone (PVP) before injection could play a key role in increasing fertilization and blastocyst formation and 2) the embryo developmental ability and blastocyst production of porcine immature oocytes retrieved after vitrification-warming and co-cultured with granulosa cells during IVM, using different fertilization techniques: in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and conventional ICSI with hyaluronic acid (HA) sperm selection, known as physiological intracytoplasmic sperm injection (PICSI) and. RESULTS: Sperm selected with HA-PICSI displayed a higher percentage of live/acrosome reacted status compared to those in control and exposed to PVP. Higher dead/acrosome reacted rates were obtained after PVP exposure compared to control and HA. In oocytes, viability significantly decreased after IVM in vitrified oocytes. Besides, IVM rates were not different between control denuded oocytes cultured with granulosa cells (DO-GC) and vitrified oocytes. Regarding fertilization parameters, IVF showed higher percentages of total fertilization rate than those obtained by ICSI and PICSI. However, results demonstrate that PICSI fertilization increased the blastocysts formation rate in control DO-GC and vitrified oocytes compared to IVF and ICSI. CONCLUSIONS: To achieve high blastocyst formation rates from vitrified GV oocytes, it is recommended that sperm should be selected with HA instead of PVP before injection since high viability and acrosome reaction rates were obtained. Also, PICSI fertilization was the best method to produce higher blastocyst rates compared to the IVF and ICSI procedures.

RESUMO

This study was designed to evaluate the effects of vitrification on immature porcine and ovine oocytes, collected at a slaughterhouse, by performing vitrification in devices with different volumes. Viability was evaluated both before and after vitrification and maturation. Immediately after warming, the percentage of viable pig oocytes was 81% regardless the type of device, while in the control (after oocyte selection) was 95%. The viability of matured pig oocytes after warming, vitrified in beveled edge open straws (BES) was 6%, in small-open-pulled-straw (SOPS) was 17% and in cryotop was 4%, while the viability of the control group was 86%. The viability and maturation results were similar with all devices. Embryo development (ED) was observed in fresh porcine oocytes with 15% 2-8 cell embryos, 7% morulae and 3% blastocysts, and non-embryo cleavage was observed in warmed oocytes. The viability of sheep oocytes immediately after warming averaged 90% in all devices, while that of the control (after oocyte selection) averaged 95%. The viability of warmed oocytes after maturation was: BES 21%, SOPS 30%, cryotop 21% and control group 86%; while maturation values were 11, 21, 34 and 70%, respectively. After vitrification, the highest ED was achieved with ovine oocytes vitrified in SOPS, with 17% morulae development and it was the only device in which blastocysts developed. A direct relationship was observed between viability and actin filament integrity in both species.

Assuntos