Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 2.003
Filtrar
1.
Zygote ; 31(1): 14-24, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36683392

RESUMO

This study investigated the effect of the flavonoid-based compound isorhamnetin (ISO) on maturation and developmental competence in oxidative stress-exposed porcine oocytes in vitro. Treatment with 2 µM ISO (2 ISO) increases the developmental rate of oxidative stress-exposed porcine oocytes during in vitro maturation (IVM). The glutathione level and mRNA expression of antioxidant-related genes (NFE2L2 and SOD2) were increased in the 2 ISO-treated group, whereas the reactive oxygen species level was decreased. Treatment with 2 ISO increased mRNA expression of a cumulus cell expansion-related gene (SHAS2) and improved chromosomal alignment. mRNA expression of maternal genes (CCNB1, MOS, BMP15 and GDF9) and mitogen activated protein kinase (MAPK) activity were increased in the 2 ISO-treated group. The total cell number per blastocyst and percentage of apoptotic cells were increased and decreased in the 2 ISO-treated group, respectively. Treatment with 2 ISO increased mRNA expression of development-related genes (SOX2, NANOG, and POU5F1) and anti-apoptotic genes (BCL2L1 and BIRC5) and decreased that of pro-apoptotic genes (CASP3 and FAS). These results demonstrate that 2 ISO improves the quality of porcine oocytes by protecting them against oxidative stress during IVM and enhances subsequent embryo development in vitro. Therefore, we propose that ISO is a useful supplement for IVM of porcine oocytes.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos , Suínos , Animais , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/fisiologia , Desenvolvimento Embrionário , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Blastocisto/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
2.
Molecules ; 28(2)2023 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-36677917

RESUMO

Galectin-1 has been cited as a mediator involved in preventing early embryonic death in mammals and is implicated in maternal-fetal tolerance. Galectin-1 is also a reasonable tool to improve fertility in assisted reproduction procedures. As recommended in the ICH guidelines (S5-R2 and S6-R1) and based on bioethical concerns, we chose bovine embryos (BE) to assess in vitro embryo development as part of a larger reproductive safety and toxicology study in progress. The design considered in vitro embryo development using rHGAL-1 supplementations (in three different concentrations) of the in vitro embryo culture (IVP) media. Based on procedures for the commercial in vitro production of BE using oocytes aspirated from slaughterhouse ovaries, rHGAL-1 supplementation was performed in two experiments: In Experiment 1 on oocyte maturation, involving IVM medium supplementation, and in Experiment 2 on culture step IVC, involving supplementation with an SOF medium. IVP commercial procedures were used, with three IVP replicates per experiment, and the oocytes we distributed into four groups of treatment (one control group and three different dosages of rHGAL-1 to supplement both IVM and SOF media using 2, 20, and 40 µg·mL-1, respectively. A total of 967 (Experiment 1) and 1213 (Experiment 2) oocytes were aspirated and submitted to the IVP procedure. There was no damage to the in vitro bovine embryo growth when considering cleavage percentage (%CLE), blastocyst development (Bl, Bx, Bh, and B) at Days 7 and 8, or an amount of rHGAL-1 supplementation ≤20 µg·mL-1. The immunohistochemistry assay with D8 embryos cultivated using rHGAL-1 supplementation on the culture medium (SOF medium) demonstrated the presence of exogenous GAL-1 distributed in mass cell and trophoblastic cells, and the profile observed was dependent on exogenous supplementation, which was most evident in hatched embryos. The findings confirmed the use of a reasonable amount of rHGAL-1 for in vitro embryonic development and would make the use of rHGAL-1 in assisted reproduction in humans more reliable and safer. Even though it was not the objective of the study, we verified that supplementation with 2 µg·mL-1 significantly improved some of the evaluated parameters of embryonic development (%BlD7, %BD7, %BlD8, %BhD8, and %BD8).


Assuntos
Galectina 1 , Técnicas de Maturação in Vitro de Oócitos , Animais , Bovinos , Feminino , Humanos , Gravidez , Embrião de Mamíferos , Desenvolvimento Embrionário , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos
3.
Theriogenology ; 198: 241-249, 2023 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-36621133

RESUMO

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/µL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/µL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.


Assuntos
Proteína Morfogenética Óssea 15 , Oócitos , Suínos , Feminino , Animais , Proteína Morfogenética Óssea 15/genética , Microinjeções/veterinária , Oócitos/fisiologia , Técnicas de Maturação in Vitro de Oócitos/veterinária , Células do Cúmulo/fisiologia , Mamíferos
4.
Eur J Obstet Gynecol Reprod Biol ; 281: 87-91, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36586210

RESUMO

OBJECTIVE(S): In vitro maturation (IVM) of oocytes retrieved ex vivo from ovarian tissue (OTO-IVM) could be an additional source of mature oocytes with the potential to optimise medical fertility preservation (FP) after oophorectomy. It is often undertaken at the same time as the ovarian tissue cryopreservation (OTC). In the presence of an organic ovarian cyst, OTO-IVM could prove to be the only technique available to permit FP since ovarian stimulation, transvaginal ovarian needle puncture or future ovarian tissue graft are contraindicated. However, the presence of an organic cyst could alter follicular growth and the number of retrievd oocytes. Our study aims to assess the efficiency of OTO-IVM in such situations. STUDY DESIGN: Retrospective, observational study involving 20 female patients with FP by OTO-IVM between May 2017 and November 2021 at the University Hospital of Toulouse. Oocytes retrieved "ex vivo" were transferred to an IVM medium with HP-hMG, LH and HSA and then vitrified after 24 to 48 h of IVM. Data analysis was performed on the total population and comparatively between patients who had or did not have an organic ovarian cyst. RESULTS: The indications included 15 oncologic and 5 non-oncologic indications. Ten had an organic ovarian cyst on the retrieved ovary. The number of retrieved oocytes was 17.4+/-12.0 in the absence of cyst vs 4.1+/-6.3 in the presence (p = 0.003). The number of vitrified mature oocytes was 5.8+/-5.3 in the absence vs 1.1+/-2.2 (median = 0) in the presence of a cyst (p = 0.03). Ninety percent of the patients with an organic cyst had less than two vitrified mature oocytes. The mean maturation rate was 34%, not significantly different between the two groups. We found a correlation between serum AMH level and the number of mature oocytes: ρ:0.47 CI95 = [0.02; 0.76]; p = 0.04. CONCLUSION(S): OTO-IVM is an additional source of mature oocytes to optimise FP after oophorectomy. However, in the presence of an organic ovarian cyst on the retrieved ovary, the exocrine, paracrine and endocrine functions of the ovary are impaired. As such, the number of immature oocytes obtained is highly impacted and appears to be insufficient to be able to propose systematically this technique in such situations.


Assuntos
Cistos , Preservação da Fertilidade , Cistos Ovarianos , Humanos , Feminino , Preservação da Fertilidade/métodos , Técnicas de Maturação in Vitro de Oócitos/métodos , Estudos Retrospectivos , Oócitos/fisiologia , Criopreservação/métodos , Cistos Ovarianos/cirurgia
5.
Reprod Toxicol ; 115: 85-93, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36549457

RESUMO

Lipopolysaccharide (LPS), a significant virulence factor of gram-negative bacteria, adversely affects female reproduction, especially the maturation and early embryonic development of oocytes, through inducing of inflammatory and oxidative stress-associated toxic responses. Resveratrol (Res), a potent antioxidant, exhibits many beneficial effects on the maturation and developmental competence of oocytes. However, it is unclear whether Res can restore LPS-induced defects in the maturation of oocytes during meiosis. In this study, we used porcine oocytes to explore the protective effects of Res and its underlying mechanism against the toxic impacts of LPS exposure on meiotic maturation and developmental competence of oocytes during meiosis. The oocytes were randomly assigned to a control, LPS-exposed or Res-supplemented group. Nuclear and cytoplasmic maturation was assessed after 26 h (MI) or 44 h (MII) of in vitro maturation (IVM). Our results showed that 10 µM Res significantly improved the rates of oocyte maturation and blastocyst formation after exposure to 15 µg/mL LPS. In addition, Res preserved the normal spindle/chromosome structure and maintained acetylated tubulin levels, actin polymerization and cortical granules (CGs) distribution. Additionally, Res protected mitochondrial content and function, scavenges reactive oxygen species (ROS), and reduced DNA damage and apoptosis in LPS-exposed oocytes. Furthermore, inhibition of SIRT1 by its specific inhibitor EX527 suppressed the recovery of ROS levels, mitochondrial content, and spindle/chromosome structure by Res supplementation. In summary, this study shows that Res can alleviate the impacts of LPS-induced toxicity on meiosis in porcine oocytes by upregulating SIRT1, which ameliorates oxidative stress and increasing mitochondrial content.


Assuntos
Lipopolissacarídeos , Sirtuína 1 , Gravidez , Suínos , Feminino , Animais , Lipopolissacarídeos/toxicidade , Resveratrol/farmacologia , Espécies Reativas de Oxigênio , Oócitos , Técnicas de Maturação in Vitro de Oócitos
6.
Hum Reprod ; 37(12): 2867-2884, 2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36342870

RESUMO

STUDY QUESTION: Can diet normalization or a calorie-restricted diet for 2 or 4 weeks be used as a preconception care intervention (PCCI) in Western-type diet-induced obese Swiss mice to restore metabolic health and oocyte quality? SUMMARY ANSWER: Metabolic health and oocyte developmental competence was already significantly improved in the calorie-restricted group after 2 weeks, while obese mice that underwent diet normalization showed improved metabolic health after 2 weeks and improved oocyte quality after 4 weeks. WHAT IS KNOWN ALREADY: Maternal obesity is linked with reduced metabolic health and oocyte quality; therefore, infertile obese women are advised to lose weight before conception to increase pregnancy chances. However, as there are no univocal guidelines and the specific impact on oocyte quality is not known, strategically designed studies are needed to provide fundamental insights in the importance of the type and duration of the dietary weight loss strategy for preconception metabolic health and oocyte quality. STUDY DESIGN, SIZE, DURATION: Outbred female Swiss mice were fed a control (CTRL) or high-fat/high-sugar (HF/HS) diet. After 7 weeks, some of the HF mice were put on two different PCCIs, resulting in four treatment groups: (i) only control diet for up to 11 weeks (CTRL_CTRL), (ii) only HF diet for up to 11 weeks (HF_HF), (iii) switch at 7 weeks from an HF to an ad libitum control diet (HF_CTRL) and (iv) switch at 7 weeks from an HF to a 30% calorie-restricted control diet (HF_CR) for 2 or 4 weeks. Metabolic health and oocyte quality were assessed at 2 and 4 weeks after the start of the intervention (n = 8 mice/treatment/time point). PARTICIPANTS/MATERIALS, SETTING, METHODS: Changes in body weight were recorded. To study the impact on metabolic health, serum insulin, glucose, triglycerides, total cholesterol and alanine aminotransferase concentrations were measured, and glucose tolerance and insulin sensitivity were analyzed at PCCI Weeks 2 and 4. The quality of in vivo matured oocytes was evaluated by assessing intracellular lipid droplet content, mitochondrial activity and localization of active mitochondria, mitochondrial ultrastructure, cumulus cell targeted gene expression and oocyte in vitro developmental competence. MAIN RESULTS AND THE ROLE OF CHANCE: Significant negative effects of an HF/HS diet on metabolic health and oocyte quality were confirmed (P < 0.05). HF_CTRL mice already showed restored body weight, serum lipid profile and glucose tolerance, similar to the CTRL_CTRL group after only 2 weeks of PCCI (P < 0.05 compared with HF_HF) while insulin sensitivity was not improved. Oocyte lipid droplet volume was reduced at PCCI Week 2 (P < 0.05 compared with HF_HF), while mitochondrial localization and activity were still aberrant. At PCCI Week 4, oocytes from HF_CTRL mice displayed significantly fewer mitochondrial ultrastructural abnormalities and improved mitochondrial activity (P < 0.05), while lipid content was again elevated. The in vitro developmental capacity of the oocytes was improved but did not reach the levels of the CTRL_CTRL mice. HF_CR mice completely restored cholesterol concentrations and insulin sensitivity already after 2 weeks. Other metabolic health parameters were only restored after 4 weeks of intervention with clear signs of fasting hypoglycemia. Although all mitochondrial parameters in HF_CR oocytes stayed aberrant, oocyte developmental competence in vitro was completely restored already after 2 weeks of intervention. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: In this study, we applied a relevant HF/HS Western-type diet to induce obesity in an outbred mouse model. Nevertheless, physiological differences should be considered when translating these results to the human setting. However, the in-depth study and follow-up of the metabolic health changes together with the strategic implementation of specific PCCI intervals (2 and 4 weeks) related to the duration of the mouse folliculogenesis (3 weeks), should aid in the extrapolation of our findings to the human setting. WIDER IMPLICATIONS OF THE FINDINGS: Our study results with a specific focus on oocyte quality provide important fundamental insights to be considered when developing preconception care guidelines for obese metabolically compromised women wishing to become pregnant. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the Flemish Research Fund (FWO-SB grant 1S25020N and FWO project G038619N). The authors declare there are no conflicts of interest.


Assuntos
Infertilidade Feminina , Insulinas , Feminino , Camundongos , Humanos , Gravidez , Animais , Técnicas de Maturação in Vitro de Oócitos/métodos , Camundongos Obesos , Restrição Calórica , Cuidado Pré-Concepcional , Oócitos/metabolismo , Infertilidade Feminina/metabolismo , Obesidade/terapia , Obesidade/metabolismo , Colesterol , Glucose , Insulinas/metabolismo , Insulinas/farmacologia , Lipídeos
7.
Biochem Biophys Res Commun ; 636(Pt 1): 155-161, 2022 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-36334439

RESUMO

BACKGROUND: Peroxiredoxin 4 (Prdx4) in the endoplasmic reticulum (ER) is the only secretory member of the antioxidant Prdx family. Our previous studies demonstrated that Prdx4 in cumulus cells (CCs) ameliorated the maturation of oocytes in vitro and enhanced oocyte developmental competence by preventing CCs apoptosis caused by oxidative stress (OS) through gap junctions. In this study, we aimed to determine whether Prdx4 released by CCs can repair meiotic defects in mouse oocytes by co-culturing immature (germinal vesicle) oocytes with CCs from mature oocytes in the absence of gap junctions. RESULTS: The OS-induced meiotic defects in mouse oocytes were impeded by co-culture with CCs, as evidenced by the increased first polar body (PB1) extrusion rate and decreased ROS level. CCs increased Prdx4 expression and lowered IRE1α, Bip expression in H2O2-treated oocytes. After knockdown of Prdx4 expression in CCs, the rate of PB1 extrusion in the oocytes was significantly reduced to the level detected in H2O2 group, and ER stress was not alleviated. CO-IP and immunofluorescence co-localization experiments demonstrated that Prdx4 interacted with PDIA6 in the oocytes and the Pearson's R value was 0.69 calculated using ImageJ. CONCLUSIONS: Cumulus cells can promote the maturation of oocytes in vitro by secreting Prdx4 in a paracrine manner and serve as a promising therapeutic antioxidant for improving the quality of oocytes, especially aging oocytes, in clinical in vitro maturation (IVM).


Assuntos
Células do Cúmulo , Técnicas de Maturação in Vitro de Oócitos , Peroxirredoxinas , Animais , Feminino , Camundongos , Antioxidantes/metabolismo , Endorribonucleases/metabolismo , Peróxido de Hidrogênio/metabolismo , Oócitos/metabolismo , Peroxirredoxinas/genética , Peroxirredoxinas/metabolismo , Proteínas Serina-Treonina Quinases
8.
Reprod Domest Anim ; 57(11): 1440-1449, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36317481

RESUMO

Increased palmitic acid (PA) levels have been found in females with reduced fertility due to metabolic disorders. However, effective antioxidant astaxanthin (AXE) might positively affect animal reproduction. Therefore, the present study was designed to evaluate the impact of a high concentration of PA on oocyte maturation and the possible protective effect of AXE against high PA concentration in pigs. Firstly, different concentrations (0.2, 0.5, 0.8 mM) of PA were conducted on in vitro maturation (IVM) of pig oocytes (PA0.2, PA0.5, and PA0.8), while no addition of PA was performed as the control group (Ctrl). Results showed that the cumulus cell expansion index (CCEI) was lower in PA0.5 and PA0.8 groups compared to Ctrl group (p < .05). In PA0.5 group, not only did the percentage of matured oocytes with the first polar body (PB1) reduced, that with more oocytes arrested at germinal vesicle (GV) stage (53.44% ± 7.16% vs. 20.93% ± 5.16%, p < .05), but also a higher number of transzonal projections (TZPs) was observed in PA0.5 than Ctrl group. Besides, supplement of PA resulted in a dose-dependent decrease in mitochondrial activity. Although no difference of lipid content was observed between PA0.5 and Ctrl groups, the lipid content was at a higher level in PA0.2 group than in the other three groups. Hence, concentration of 0.5 mM of PA was performed in the following experiments, and 2.5 µM AXE carried out to investigate the possible relief effects of PA (PA0.5 + AXE). Results showed that the percentage of matured oocytes with PB1 was higher in PA0.5 + AXE than in PA0.5 group (63.43% ± 1.50% vs. 55.33% ± 0.81%, p < .01), and ROS levels both in oocytes and their cumulus cells (CCs) reduced in PA0.5 + AXE when compared to PA0.5 group. In addition, the rate of CCs with apoptosis decreased in PA0.5 + AXE, and the expression level of caspase 3 and BAX was lower than PA0.5 group. In conclusion, the maturation of pig oocytes was inhibited by the high concentration of PA; however, this negative effect of PA-induced might be relieved by the supplement of AXE.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Ácido Palmítico , Feminino , Animais , Suínos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos , Ácido Palmítico/farmacologia , Ácido Palmítico/metabolismo , Células do Cúmulo , Oócitos
9.
Aging (Albany NY) ; 14(22): 9200-9209, 2022 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-36441531

RESUMO

Women over age 35 suffer from the inadequate number and poor quality of oocytes during assisted reproductive treatment, and making full use of the oocytes by in vitro maturation (IVM) is crucial. Rapamycin could improve the developmental competences of the post-maturation oocytes during in vitro aging, yet its effects on the IVM process of oocytes from an aged population were not clear. In this study, the immature oocytes from aged mice or older women underwent IVM with or without 10 nM rapamycin, followed by parthenogenetic activation or insemination and embryo culture. The developmental competence and quality of IVM oocytes in both groups were compared. The results showed that in aged mice, the maturation rate, activation rate, and cleavage rate of IVM oocytes were significantly elevated in the rapamycin group. Additionally, oocytes cultured with rapamycin presented decreased ROS levels, reduced chromosome aberration, and attenuated levels of γ-H2AX. During IVM of oocytes from older women, the GVBD rate, 24 h maturation rate, and 48 h maturation rate were increased in the rapamycin group, compared with those in the control group, although without significant differences. After intracytoplasmic sperm injection (ICSI) and further culture of human oocytes, the high-quality embryo rate in the rapamycin group was significantly elevated. Overall, rapamycin improved IVM outcomes of oocytes from aged mice and older women. The specific mechanism of the positive effects of rapamycin on IVM outcomes might be reducing ROS levels, mitigating DNA damage, and promoting developmental potential.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Sirolimo , Humanos , Masculino , Feminino , Animais , Camundongos , Idoso , Técnicas de Maturação in Vitro de Oócitos/métodos , Espécies Reativas de Oxigênio , Sirolimo/farmacologia , Sêmen , Oócitos
10.
Cells ; 11(22)2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36429039

RESUMO

In conventional assisted reproductive technologies (ARTs), oocytes are in vitro cultured in static conditions. Instead, dynamic systems could better mimic the physiological in vivo environment. In this study, a millifluidic in vitro oocyte maturation (mIVM) system, in a transparent bioreactor integrated with 3D printed supports, was investigated and modeled thanks to computational fluid dynamic (CFD) and oxygen convection-reaction-diffusion (CRD) models. Cumulus-oocyte complexes (COCs) from slaughtered lambs were cultured for 24 h under static (controls) or dynamic IVM in absence (native) or presence of 3D-printed devices with different shapes and assembly modes, with/without alginate filling. Nuclear chromatin configuration, mitochondria distribution patterns, and activity of in vitro matured oocytes were assessed. The native dynamic mIVM significantly reduced the maturation rate compared to the static group (p < 0.001) and metaphase II (MII) oocytes showed impaired mitochondria distribution (p < 0.05) and activity (p < 0.001). When COCs were included in a combination of concave+ring support, particularly with alginate filling, oocyte maturation and mitochondria pattern were preserved, and bioenergetic/oxidative status was improved (p < 0.05) compared to controls. Results were supported by computational models demonstrating that, in mIVM in biocompatible inserts, COCs were protected from shear stresses while ensuring physiological oxygen diffusion replicating the one occurring in vivo from capillaries.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Ovário , Feminino , Ovinos , Animais , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/fisiologia , Oxigênio , Alginatos/farmacologia
11.
Molecules ; 27(20)2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36296422

RESUMO

The quality of in vitro matured oocytes is inferior to that of in vivo matured oocytes, which translates to low developmental capacity of embryos derived from in vitro matured oocytes. The developmental potential of in vitro matured oocytes is usually impaired due to oxidative stress. Stromal cell-derived factor-l (SDF1) can reduce oxidative stress and inhibit apoptosis. The aim of this study was to investigate the effects of SDF1 supplementation during pig oocyte in vitro maturation (IVM) on subsequent embryo development, and to explore the acting mechanisms of SDF1 in pig oocytes. We found that the IVM medium containing 20 ng/mL SDF1 improved the maturation rate of pig oocytes, as well as the cleavage rate and blastocyst rate of embryos generated by somatic cell nuclear transfer, in vitro fertilization, and parthenogenesis. Supplementation of 20 ng/mL SDF1 during IVM decreased the ROS level, increased the mitochondrial membrane potential, and altered the expression of apoptosis-related genes in the pig oocytes. The porcine oocyte transcriptomic data showed that SDF1 addition during IVM altered the expression of genes enriched in the purine metabolism and TNF signaling pathways. SDF1 supplementation during pig oocyte IVM also upregulated the mRNA and protein levels of YY1 and TET1, two critical factors for oocyte development. In conclusion, supplementation of SDF1 during pig oocyte IVM reduces oxidative stress, changes expression of genes involved in regulating apoptosis and oocyte growth, and enhances the ability of in vitro matured pig oocytes to support subsequent embryo development. Our findings provide a theoretical basis and a new method for improving the developmental potential of pig in vitro matured oocytes.


Assuntos
Desenvolvimento Embrionário , Técnicas de Maturação in Vitro de Oócitos , Suínos , Animais , Técnicas de Maturação in Vitro de Oócitos/métodos , Espécies Reativas de Oxigênio/farmacologia , Suplementos Nutricionais , RNA Mensageiro , Purinas/farmacologia
12.
Theriogenology ; 194: 133-143, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36244270

RESUMO

Porcine in vitro fertilization often results in low embryo development rates compared to other livestock species, which is often associated with either a low fertilization rate or high incidence of polyspermy. Since the quality of oocyte maturation is known to play a significant role in oocyte competence, we investigated the impact of supplementing in vitro maturation (IVM) medium containing porcine follicular fluid (pFF) with the growth factors FGF2, LIF and IGF1 (FLI), along with different combinations of cysteine, melatonin and ITS, on cumulus cell expansion, oocyte meiotic maturation, fertilization outcome, embryo development and blastocyst cell numbers. Maturation medium containing pFF yielded the greatest cumulus expansion. Compared to pFF and FLI individually, using pFF and FLI together resulted in the best embryo development rates over total oocyte placed in IVF (12.5% vs. 15.0% vs. 26.6%, respectively). Supplementation of IVM medium containing pFF and FLI with either cysteine, melatonin or insulin-transferrin-selenium, revealed that cysteine was essential to improve embryo development, while melatonin and ITS had a limited impact on improving blastocyst rates. Finally, we observed that pig oocytes matured in medium supplemented with pFF, FLI, cysteine and melatonin had a high proportion of monospermic zygotes (68.2%) and low proportion of polyspermic zygotes (15.9%) following IVF and yielded superior cleavage (78.2%) and blastocyst (32.0%) rates.


Assuntos
Antioxidantes , Melatonina , Feminino , Animais , Suínos , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Cisteína , Melatonina/farmacologia , Melatonina/metabolismo , Oócitos , Fertilização In Vitro/veterinária , Fertilização In Vitro/métodos , Desenvolvimento Embrionário , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Suplementos Nutricionais , Técnicas de Maturação in Vitro de Oócitos/veterinária , Técnicas de Maturação in Vitro de Oócitos/métodos
13.
Theriogenology ; 193: 128-135, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36162289

RESUMO

Autophagy plays an important role in mammalian oocyte maturation and early embryonic development and rapamycin is well known for inducing autophagy. Although previous studies have reported the effects of rapamycin on oocytes in vitro maturation (IVM) in different species, few studies have been reported on the role of rapamycin in yak oocytes IVM and embryonic development. Therefore, the objective of this study was to examine the effect of rapamycin treatment on yak oocytes IVM and early embryonic development. Specifically, immature yak oocytes during IVM or parthenogenetic (PA) embryos were treated with different rapamycin concentrations to select an optimal dose. Then evaluated its effect on maturation rates, cleavage, and blastocyst formation rates, mitochondrial membrane potential, ROS levels. Related genes and proteins expression in matured oocytes and blastocysts were also evaluated. The results show that 10 nM rapamycin treatment during IVM significantly improved oocyte maturation rates of oocytes and blastocyst formation rates. Treatment with 10 nM rapamycin reduced ROS level but increased mitochondrial membrane potential. Correspondingly, mRNA and protein expressions of LC3, Beclin-1, and Bcl-2 up-regulated while Bax down-regulated in matured yak COCs. When parthenogenetic embryos were treated with different rapamycin concentrations, 10 nM rapamycin treatment showed higher 8-cell and blastocyst formation rates. Also, CDX2, POU5F1, SOX2, and Nanog levels in blastocysts were upregulated. In summary, our findings demonstrate that rapamycin treatment improves oocytes maturation probably by increasing mitochondrial membrane potential, reducing ROS levels, and regulating the apoptosis in mature yak oocytes. Rapamycin treatment also improves embryonic developmental competence in the yak.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Sirolimo , Animais , Proteína Beclina-1/metabolismo , Proteína Beclina-1/farmacologia , Blastocisto/fisiologia , Bovinos , Desenvolvimento Embrionário , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Mamíferos , Oócitos/fisiologia , Partenogênese , Gravidez , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sirolimo/metabolismo , Sirolimo/farmacologia , Proteína X Associada a bcl-2/metabolismo
14.
Reprod Fertil Dev ; 34(17): 1089-1098, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36162418

RESUMO

CONTEXT: In vitro maturation (IVM) of oocytes is an alternative approach for patients with polycystic ovary syndrome (PCOS) predisposing to ovarian hyperstimulation syndrome (OHSS). Transcriptomic analysis of cumulus cells (CC) may help make IVM more efficient. The aim of this study was to examine the impact of miR-144 and miR-224 and their candidate target genes (COX-2 and PTX-3 , respectively) expression on oocyte development in PCOS patients. METHODS: Immature oocytes were retrieved from 20 PCOS patients. After IVM, samples were divided into two groups: matured (M) and immatured (I) oocytes. ICSI was performed and the embryo quality was evaluated. qPCR was used to analyse miR-144, miR-224, COX-2 and PTX-3 expression levels in CCs of each group. KEY RESULTS: We found that the expression levels of miR-144 and miR-224 were lower and the COX-2 and PTX-3 mRNA levels were higher in CCs of M group than in CCs of I group. The expression level of miR-144 and miR-224 in unfertilised oocytes were higher than fertilised oocytes. The contrary results were observed for COX-2 and PTX-3 . A reduction pattern in the expression level of miR-144 and miR-224 and increasing pattern in the level of COX-2 and PTX-3 expression were observed in high quality compared to low quality embryos. CONCLUSIONS: The selected miRNAs were related to oocyte maturation, fertilisation and embryo development. These results support their critical involvement in oocyte development. IMPLICATIONS: Our findings may help reveal the mechanisms of post-transcriptional regulation by miR-144 and miR-224 during IVM procedure.


Assuntos
MicroRNAs , Síndrome do Ovário Policístico , Humanos , Feminino , Técnicas de Maturação in Vitro de Oócitos/métodos , Células do Cúmulo/metabolismo , Síndrome do Ovário Policístico/genética , Síndrome do Ovário Policístico/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Oócitos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Fertilização
15.
Anim Reprod Sci ; 245: 107071, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36152450

RESUMO

The demand for equine in vitro produced embryos has increased over the last decade. The aim of this study was to compare the effects of an extended IVM or a prolonged period before fertilization, including holding time, on equine immature oocyte developmental competence. Oocytes, collected from abattoir-derived ovaries, were divided into 4 groups: H0/24 (n = 165) 0 h holding + standard 24-26 h IVM; H8/36 (n = 160) 8 h holding + 36 h IVM; H20/24 (n = 187) 20 h holding + 24 h IVM; H0/44 (n = 164) 0 h holding + 44 h IVM. Oocytes matured to MII were fertilized by intracytoplasmic sperm injection (ICSI) and cultured for 10 days. The oocyte degeneration rate was higher (P < 0.05) for H20/24 than the other groups (H0/24 38.2 %, H8/36 43.1 %, H20/24 54.5 %, H0/44 32.9 %). Cleavage was higher (P < 0.05) in H20/24 (70 %) compared to H0/24 (45 %) and H8/36 (54 %) but not to H0/44 (63 %). No differences among groups were observed in the number of blastocysts per oocyte. Injected oocytes that reached the blastocysts stage were higher (P < 0.05) for H20/24 (20 %) than H0/24 (7 %) and H0/44 (7 %) but not H8/36 (12 %). For cleaved oocytes, a higher blastocyst rate (P < 0.05) was observed for H20/24 (28 %) than H0/44 (11 %), while H0/24 (15 %) and H8/36 (21 %) were not different from any group (P > 0.05). Timing of blastocyst development was not different among groups. Overnight holding of equine immature oocytes followed by a standard IVM interval may induce a pre-selection of the most competent oocytes thereby improving cleavage and embryo development rates after ICSI.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Sêmen , Animais , Blastocisto , Desenvolvimento Embrionário , Cavalos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Oócitos , Injeções de Esperma Intracitoplásmicas/veterinária
16.
Zygote ; 30(6): 882-890, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36148786

RESUMO

This study aims to evaluate the effects of N-acetylcysteine (NAC) on bovine oocyte maturation, mitochondrial activity and transzonal projections (TZP), as well as on the levels of reactive oxygen species (ROS) and messenger RNA (mRNA) for catalase (CAT) superoxide dismutase (SOD), periredoxin-6 (Prdx6), glutathione peroxidase (GPx), growth and differentiation factor-9 (GDF9), histone H1Foo, cyclin B1 (CCNB1) and c-Mos. Bovine cumulus-oocyte complexes (COC) of medium-sized antral follicles (3.0-6.0 mm) were prematured in TCM-199 for 8 h at 38.5°C in 5% CO2. After prematuration in the presence of forskolin and C-type natriuretic peptide, COCs were matured in TCM-199 alone or with 0.1, 0.5 or 2.5 mM NAC. Then, oocytes were classified according to the stage of chromatin. Furthermore, mitochondrial activity and intracellular levels of ROS and TZP were also evaluated. The levels of mRNAs for CAT, SOD, Prdx6, GPx, GDF9, H1Foo, CCNB1 and c-Mos were evaluated using real-time polymerase chain reaction (RT-PCR). The results showed that NAC significantly increased the percentages of oocytes with resumption of meiosis when compared with those oocytes matured in control medium. Oocytes had homogeneous mitochondrial distribution, and those cultured with 0.1 and 0.5 mM NAC had lower levels of ROS when compared with the control. In addition, 0.5 mM NAC reduced TZP and the levels of mRNA for CCNB1. In contrast, NAC did not influence the expression of CAT, GPx, Prdx6, SOD, GDF9, H1Foo, and c-Mos. In conclusion, 0.5 mM NAC reduced the levels of ROS, TZP and mRNA for CCNB1, and improved in vitro resumption of meiosis in oocytes from medium-sized bovine antral follicles.


Assuntos
Acetilcisteína , Técnicas de Maturação in Vitro de Oócitos , Bovinos , Animais , Técnicas de Maturação in Vitro de Oócitos/métodos , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Acetilcisteína/metabolismo , Oócitos , Meiose , Superóxido Dismutase/metabolismo , Glutationa Peroxidase/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
17.
Zygote ; 30(6): 854-862, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36106341

RESUMO

Heat shock protein 90 (Hsp90) is critical for cell homeostasis but its role on bovine oocyte maturation is not well known. We investigated the importance of Hsp90 for competence of bovine oocyte using 17-(allylamino)-17-demethoxygeldanamycin (17AAG), an inhibitor of Hsp90, during in vitro maturation (IVM). Three experiments evaluated the effect of 17AAG on developmental competence of oocytes matured in vitro under thermoneutral (38.5ºC) or heat shock (HS; 41.5ºC) temperatures. The first experiment found that the blastocyst rates were lower (P < 0.05) with 2 µM 17AAG compared with the untreated control (0 µM). The abundance of HSF1 transcripts was higher in oocytes matured with 2 µM than with 0 and 1 µM 17AAG, whereas the abundance of HSP90AA1 and HSPA1A transcripts was lower (P < 0.05) with 1 and 2 µM than with 0 µM. The second experiment found that 2 µM 17AAG for 12 or 24 h during IVM decreased (P < 0.05) the blastocysts rates. In the third experiment, the association of 2 µM 17AAG with HS for 12 h during IVM resulted in lower (P < 0.05) blastocysts rates than 17AAG, HS or untreated control. In conclusion, inhibition of Hsp90 during in vitro maturation compromises further embryo development; the association of Hsp90 inhibition with HS aggravates the deleterious effect of both on oocyte developmental competence.


Assuntos
Técnicas de Maturação in Vitro de Oócitos , Oócitos , Bovinos , Animais , Técnicas de Maturação in Vitro de Oócitos/métodos , Oócitos/fisiologia , Lactamas Macrocíclicas/farmacologia , Lactamas Macrocíclicas/metabolismo , Resposta ao Choque Térmico , Blastocisto/fisiologia , Proteínas de Choque Térmico HSP90/genética
18.
Theriogenology ; 192: 109-115, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36113319

RESUMO

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.


Assuntos
Eugenol , Técnicas de Maturação in Vitro de Oócitos , Animais , Antioxidantes/farmacologia , Blastocisto , Calreticulina , Bovinos , Contagem de Células/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária
19.
Theriogenology ; 192: 62-72, 2022 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-36063671

RESUMO

Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 µM), and the effect of cortisol addition (0.25 µM, 0.5 µM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1.


Assuntos
Células do Cúmulo , Vitrificação , Animais , Apoptose , Bovinos , Células do Cúmulo/fisiologia , Suplementos Nutricionais , Feminino , Glucocorticoides/metabolismo , Glucocorticoides/farmacologia , Hidrocortisona/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Hidroxiesteroide Desidrogenases/farmacologia , Imunofilinas/metabolismo , Imunofilinas/farmacologia , Técnicas de Maturação in Vitro de Oócitos/métodos , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oócitos/fisiologia , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Receptores de Mineralocorticoides/metabolismo , Fatores de Transcrição/metabolismo , Ubiquinona/análogos & derivados , Proteína X Associada a bcl-2/metabolismo
20.
Reprod Biol Endocrinol ; 20(1): 133, 2022 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-36056438

RESUMO

BACKGROUND: The serum TSH level of PCOS patients was higher than that of the general female population. For patients with thyroid dysfunction, the abnormal TSH level is negatively related to the outcomes of assisted reproductive technology, but for PCOS patients with normal thyroid function, the effect of TSH level on outcomes of in vitro fertilization has not been reported. In this study, PCOS patients with normal thyroid function were included in this study to evaluate the effect of TSH on the outcomes of IVF-ET. METHODS: A retrospective cohort study was conducted to analyze the clinical data of 3190 patients who underwent IVF-ET in the Department of Human Reproductive Center of Renmin Hospital Hubei University of Medicine from January 2017 to July 2021, including 594 PCOS patients and 2595 non PCOS patients. The IVF-ET outcomes between the two groups were compared; Multi-factor linear regression analysis was used to analyze the correlation between the related variables and the oocyte maturation of PCOS patients; The ROC curve of the effect of TSH on oocyte maturation in PCOS patients was drawn. The PCOS patients were divided into TSH < 2.98 group (n = 454) and TSH ≥ 2.98 group (n = 141) according to ROC threshold TSH 2.98, and the outcomes were compared. RESULTS: TSH level in PCOS group was significantly higher than that in non-PCOS group (2.42 ± 0.86 vs 2.00 ± 0.89 UU / ml, P < 0.01), and the oocyte maturation rate and 2PN fertilization rate in PCOS group were lower than those in non-PCOS group (90.9% vs 92.4%, P = 0.02) (84.57% vs 86.77%, P = 0.02). There was no significant difference in cleavage rate and high-quality embryo rate between the two groups (P > 0.05); There was no difference in clinical pregnancy rate, abortion rate, ectopic pregnancy rate and live birth rate between the two groups (P > 0.05). Multi-factor linear regression analysis showed that TSH was negatively correlated with oocyte maturation in PCOS patients [ß = -0.124, P = 0.013,95%CI (-0.027 ~ -0.003)]; The oocyte maturation rate in TSH < 2.98 group was significantly higher than that in TSH ≥ 2.98 group (91.7% vs 88.2%, P = 0.001). CONCLUSION: The TSH level of PCOS patients with normal thyroid function is higher than that of normal people, and it is negatively correlated with the oocyte maturation in in-vitro fertilization. The ROC curve showed that when TSH > 2.98uIU/ml, the possibility of immature oocytes was higher (specificity 28.9%, sensitivity 83.0%).


Assuntos
Síndrome do Ovário Policístico , Glândula Tireoide , Feminino , Fertilização In Vitro , Humanos , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Gravidez , Taxa de Gravidez , Técnicas de Reprodução Assistida , Estudos Retrospectivos , Tireotropina
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...