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1.
Reprod Domest Anim ; 59(5): e14595, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38773768

RESUMEN

Oocyte maturation involves both nuclear and cytoplasmic maturation. Mogroside V (MV) has been shown to enhance nuclear maturation, mitochondrial content, and developmental potential of porcine oocyte during in vitro maturation (IVM). However, the impact of MV on cytoplasmic maturation and its underlying mechanisms are not understood. This study aimed to assess the effect of MV on cytoplasmic maturation. Germinal vesicle (GV) oocytes treated with MV exhibited a noticeable increase in cortical granules (CGs) formation. Additionally, MV enhanced the expression of NNAT and improved glucose uptake in mature oocytes. Further insights were gained through Smart-seq2 analysis of RNA isolated from 100 oocytes. A total of 11,274 and 11,185 transcripts were identified in oocytes treated with and without MV, respectively. Among quantified genes, 438 differentially expressed genes (DEGs) were identified for further analysis. Gene Ontology (GO) enrichment analysis indicated that these DEGs were primarily involved in DNA repair regulation, cellular response to DNA damage, intracellular components, and organelles. Furthermore, the DEGs were significantly enriched in three KEGG pathways: fatty acid synthesis, pyruvate metabolism, and WNT signalling. To validate the results, lipid droplets (LD) and triglyceride (TG) were examined. MV led to an increase in the accumulation of LD and TG production in mature oocytes. These findings suggest that MV enhances cytoplasmic maturation by promoting lipid droplet synthesis. Overall, this study provides valuable insights into the mechanisms through which MV improves oocyte quality during IVM. The results have significant implications for research in livestock reproduction and offer guidance for future studies in this field.


Asunto(s)
Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Animales , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos/efectos de los fármacos , Femenino , Porcinos , Gotas Lipídicas/metabolismo , Diterpenos/farmacología , Triglicéridos/metabolismo , Triterpenos
2.
Theriogenology ; 223: 11-21, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38657435

RESUMEN

Various models have been established to culture whole follicles of the Preantral stage; however, the process remains inefficient and is an ongoing challenge formation. It is reported that oocyte-cumulus-granulosa complexes (OCGCs) isolated from Early Antral follicles (EAFs) undergo in vitro growth (IVG) and acquire meiotic competence in some animals. However, IVG for the oocyte-granulosa complexes (OGCs) from Preantral Follicles (PAFs) has not been firmly established. The present study indicated that the use of a modified medium with Ascorbic Acid (50 µM) facilitated granulosa cell proliferation, promoted cumulus cell differentiations, and increased antrum formation for the OGCs isolated from PAFs (0.3-0.4 mm). However, the two-dimensional 96-well plate system (2D) experienced smaller size follicles and could not prolong more than 10 days of IVG. Another method is to use an Agarose matrix 3D system to provide a soft, non-adhesive base that supports the IVG of OGCs isolated from PAFs and promotes cell proliferation, antrum formation, and maintenance for 14 days. OGCs that were grown using this method retained their spherical morphology, which in turn helped to attain healthy granulosa cells and maintain their connection with oocytes, in addition, these oocytes significantly increased diameter and lipid content, indicating developmental competence. Our result indicated that the OGCs from PAFs after IVG undergo a change in chromatin morphology and expression of acetylation of histone H3 at lysine 9 (Ac-H3-K9) and methylation of histone H3 at lysine 4 (Me-H3-K4), similar to the in vivo oocytes isolated from the ovary. Likewise, IVG oocytes cultured for maturation showed full cumulus expansion and reached mature oocytes. Furthermore, after in vitro maturation, IVG oocytes underwent the first cleavage following parthenogenetic activation. In conclusion, while most studies used whole follicles from the Preantral stage for IVG, our research finding was the first to reveal that oocytes isolated from the final stage of PAFs can migrate out of the follicle and undergo IVG under suitable conditions.


Asunto(s)
Células de la Granulosa , Oocitos , Folículo Ovárico , Sefarosa , Animales , Femenino , Folículo Ovárico/efectos de los fármacos , Porcinos , Sefarosa/química , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/veterinaria
3.
Theriogenology ; 223: 53-58, 2024 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-38678696

RESUMEN

Mares enrolled in assisted reproductive technologies (ARTs) programs are often treated with non-steroidal anti-inflammatory drugs (NSAIDs), particularly phenylbutazone (Bute), due to chronic lameness. The current study was performed to determine the effect of Bute administration on the developmental competence of in vitro-matured equine oocytes subjected to Intracytoplasmic Sperm Injection (ICSI). In a Preliminary Study, immature cumulus-oocyte complexes (COCs) recovered by post-mortem ovary harvested from two healthy mares (n = 2) treated for 10 days with Bute (4.4 mg/kg, PO, BID), and four non-treated healthy mares (n = 4), were matured in vitro and subjected to Piezo-driven ICSI. Lower oocyte in vitro maturation [Bute: 25% (3/12) vs. Control: 61% (28/46)] and blastocyst rates [Bute: 0% (0/12) vs. Control: 18% (5/28)] were observed in the Bute-treated when compared to the Control mares (P < 0.05). In the Main Experiment, a group of healthy mares (n = 9) received a daily dose of Bute (4.4 mg/kg, orally, SID) for 10 days. A control group of mares (n = 10) was treated with an equal volume of placebo. Mares in both groups were subjected to ultrasound-guided transvaginal oocyte aspiration (TVA) on days 3, 33, and 77 following the last dose of Bute (PT). Recovered COCs from both mare groups were matured in vitro and subjected to Piezo-driven ICSI. By day-3 PT, oocyte in vitro maturation rate was similar between mare groups [Bute: 65% (36/55) vs. Control: 67% (78/116); P > 0.05], while oocyte recovery [Bute: 53% (55/103) vs. Control: 70% (116/166)], cleavage [Bute: 31% (11/36) vs. Control: 62% (48/78)] and blastocyst rates [Bute: [0%] (0/36) vs. Control: 28% (22/78)] were significantly different (P < 0.05). By day 33 PT and 77 PT, differences on oocyte recovery, in vitro maturation, cleavage, and blastocyst rates were not observed between mare groups. In summary, the administration of Bute for 10 consecutive days (4.4 mg/kg, PO, SID, or BID) is associated with a decrease in the ability of immature equine oocytes to undergo in vitro-maturation (Preliminary Study) and develop to the blastocyst stage following ICSI (Preliminary Study and Main Experiment). This negative effect appeared to be transient, as 30- and 77-days post-treatment, no differences on in vitro maturation, cleavage or blastocyst rates were observed.


Asunto(s)
Antiinflamatorios no Esteroideos , Blastocisto , Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Fenilbutazona , Inyecciones de Esperma Intracitoplasmáticas , Animales , Caballos , Inyecciones de Esperma Intracitoplasmáticas/veterinaria , Inyecciones de Esperma Intracitoplasmáticas/métodos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos/efectos de los fármacos , Oocitos/fisiología , Antiinflamatorios no Esteroideos/farmacología , Antiinflamatorios no Esteroideos/administración & dosificación , Fenilbutazona/farmacología , Blastocisto/efectos de los fármacos , Desarrollo Embrionario/efectos de los fármacos
4.
Theriogenology ; 222: 31-44, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38615434

RESUMEN

There is still no consensus regarding the role of lipid modulators during in vitro embryo production. Thus, we investigated how lipid reducers during the in vitro maturation of oocytes (IVM) or in vitro culture (IVC) of embryos impact their cryotolerance. A literature search was performed using three databases, recovering 43 articles for the systematic review, comprising 75 experiments (13 performed in IVM, 62 in IVC) and testing 13 substances. In 39 % of the experiments, an increase in oocyte and/or embryo survival after cryopreservation was reported, in contrast to 48 % exhibiting no effect, 5 % causing negative effects, and 8 % influencing in a dose-dependent manner. Of the 75 experiments extracted during IVM and IVC, 41 quantified the lipid content. Of those that reduced lipid content (n = 26), 50 % increased cryotolerance, 34 % had no effect, 8 % harmed oocyte/embryo survival, and 8 % had different results depending on the concentration used. Moreover, 28 out of the 43 studies were analyzed under a meta-analytical approach at the IVC stage in cattle. There was an improvement in the cryotolerance of bovine embryos when the lipid content was reduced. Forskolin, l-carnitine, and phenazine ethosulfate positively affected cryotolerance, while conjugated linoleic acid had no effect and impaired embryonic development. Moreover, fetal bovine serum has a positive impact on cryotolerance. SOF and CR1aa IVC media improved cryotolerance, while mSOF showed no effect. In conclusion, lipid modulators did not unanimously improve cryotolerance, especially when used in IVM, but presented positive effects on cryotolerance during IVC when reaching lipid reduction.


Asunto(s)
Criopreservación , Técnicas de Cultivo de Embriones , Animales , Criopreservación/veterinaria , Criopreservación/métodos , Técnicas de Cultivo de Embriones/veterinaria , Lípidos/química , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Fertilización In Vitro/veterinaria , Bovinos/embriología , Metabolismo de los Lípidos , Embrión de Mamíferos/fisiología
5.
Theriogenology ; 222: 66-79, 2024 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-38626583

RESUMEN

In vitro maturation (IVM) and cryopreservation of goat oocytes are important for establishing a valuable genetic bank for domesticated female animals and improving livestock reproductive efficiency. C-Phycocyanin (PC) is a Spirulina extract with antioxidant, antiinflammatory, and radical scavenging properties. However, whether PC has positive effect on goat oocytes IVM or developmental competence after vitrification is still unknown. In this study, we found that first polar body extrusion (n = 293), cumulus expansion index (n = 269), and parthenogenetic blastocyst formation (n = 281) were facilitated by adding 30 µg/mL PC to the oocyte maturation medium when compared with the control groups and that supplemented with 3, 10, 100 or 300 µg/mL PC (P < 0.05). Although PC supplementation did not affect spindle formation or chromosome alignment (n = 115), it facilitated or improved cortical granules migration (n = 46, P < 0.05), mitochondria distribution (n = 39, P < 0.05), and mitochondrial membrane potential (n = 46, P < 10-4). Meanwhile, supplementation with 30 µg/mL PC in the maturation medium could significantly inhibit the reactive oxygen species accumulation (n = 65, P < 10-4), and cell apoptosis (n = 42, P < 0.05). In addition, PC increased the oocyte mRNA levels of GPX4 (P < 0.01), and decreased the mRNA and protein levels of BAX (P < 0.01). Next, we investigated the effect of PC supplementation in the vitrification solution on oocyte cryopreservation. When compared with the those equilibrate in the vitrification solution without PC, recovered oocytes in the 30 µg/mL PC group showed higher ratios of normal morphology (n = 85, P < 0.05), survival (n = 85, P < 0.05), first polar body extrusion (n = 62, P < 0.05), and parthenogenetic blastocyst formation (n = 107, P < 0.05). Meanwhile, PC supplementation of the vitrification solution increased oocyte mitochondrial membrane potential (n = 53, P < 0.05), decreased the reactive oxygen species accumulation (n = 73, P < 0.05), promoted mitochondria distribution (n = 58, P < 0.05), and inhibited apoptosis (n = 46, P < 10-3). Collectively, our findings suggest that PC improves goat oocyte IVM and vitrification by reducing oxidative stress and early apoptosis, which providing a novel strategy for livestock gamete preservation and utilization.


Asunto(s)
Criopreservación , Cabras , Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Ficocianina , Vitrificación , Animales , Oocitos/efectos de los fármacos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Vitrificación/efectos de los fármacos , Criopreservación/veterinaria , Criopreservación/métodos , Ficocianina/farmacología , Femenino , Especies Reactivas de Oxígeno/metabolismo , Potencial de la Membrana Mitocondrial/efectos de los fármacos
6.
Res Vet Sci ; 171: 105222, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38513461

RESUMEN

In vitro maturation (IVM) of oocytes is clinically used in horses to produce blastocysts but current conditions used for horses are suboptimal. We analyzed the composition of equine preovulatory follicular fluid (FF) secretome and tested its effects on meiotic competence and gene expression in oocytes subjected to IVM. Preovulatory FF was obtained, concentrated using ultrafiltration with cut-off of 10 kDa, and stored at -80 °C. The metabolic and proteomic composition was analyzed, and its ultrastructural composition was assessed by cryo-transmission microscopy. Oocytes obtained post-mortem or by ovum pick up (OPU) were subjected to IVM in the absence (control) or presence of 20 or 40 µg/ml (S20 or S40) of secretome. Oocytes were then analyzed for chromatin configuration or snap frozen for gene expression analysis. Proteomic analysis detected 255 proteins in the Equus caballus database, mostly related to the complement cascade and cholesterol metabolism. Metabolomic analysis yielded 14 metabolites and cryo-transmission electron microscopy analysis revealed the presence of extracellular vesicles (EVs). No significant differences were detected in maturation rates among treatments. However, the expression of GDF9 and BMP15 significantly increased in OPU-derived oocytes compared to post-mortem oocytes (fold increase ± SEM: 9.4 ± 0.1 vs. 1 ± 0.5 for BMP15 and 9.9 ± 0.3 vs. 1 ± 0.5 for GDF9, respectively; p < 0.05). Secretome addition increased the expression of TNFAIP6 in S40 regardless of the oocyte source. Further research is necessary to fully understand whether secretome addition influences the developmental competence of equine oocytes.


Asunto(s)
Líquido Folicular , Proteómica , Femenino , Caballos , Animales , Líquido Folicular/química , Líquido Folicular/metabolismo , Secretoma , Meiosis , Oocitos/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria
7.
Theriogenology ; 220: 84-95, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38490113

RESUMEN

Understanding the mechanisms for oocyte maturation and optimizing the protocols for in vitro maturation (IVM) are greatly important for improving developmental potential of IVM oocytes. The miRNAs expressed in cumulus cells (CCs) play important roles in oocyte maturation and may be used as markers for selection of competent oocytes/embryos. Although a recent study from our group identified several new CCs-expressed miRNAs that regulate cumulus expansion (CE) and CC apoptosis (CCA) in mouse oocytes, validation of these findings and further investigation of mechanisms of action in other model species was essential before wider applications. By using both in vitro and in vivo pig oocyte models with significant differences in CE, CCA and developmental potential, the present study validated that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes. We demonstrated that miR-149 and miR-31 targeted SMAD family member 6 (SMAD6) and transforming growth factor ß2 (TGFB2), respectively, in the transforming growth factor-ß (TGF-ß) signaling. Furthermore, both miR-149 and miR-31 increased CE and decreased CCA via activating SMAD family member 2 (SMAD2) and increasing the expression of SMAD2 and SMAD family member 4. In conclusion, the present results show that miR-149 and miR-31 improved CE and developmental potential while suppressing CCA of pig oocytes by activating the TGF-ß signaling, suggesting that they might be used as markers for pig oocyte quality.


Asunto(s)
Células del Cúmulo , Técnicas de Maduración In Vitro de los Oocitos , MicroARNs , Oocitos , Animales , Femenino , Células del Cúmulo/fisiología , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , MicroARNs/genética , MicroARNs/metabolismo , Oocitos/fisiología , Porcinos , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta/metabolismo
8.
Anim Reprod Sci ; 262: 107426, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38377631

RESUMEN

The reproductive management of the buffalo species still faces several unresolved problems, which directly affect the productivity of the herd, one of them being the presence of repeat breeder females. Given this scenario, this study aimed to verify the developmental competence of oocytes obtained from repeat breeder females and submitted to parthenogenetic activation. In addition, embryo gene expression was compared to normally fertile females. Murrah buffaloes were divided into two groups: repeat breeder (RB, n = 8) and normally fertile or control (CR, n = 7). Cumulus-oocyte complexes (COCs) were aspirated by transvaginal ovum pick-up from estrus synchronized females. The COCs were submitted to IVM for 24 h, and subsequently, the oocytes were activated using ionomycin, followed by 6-DMAP. Afterwards, the presumptive parthenotes were cultured for six or seven days in a microenvironment of 5 % CO2, 5 % O2, and 90 % N2 at 38.5 °C. The expression of OCT4, GLUT1, BCL2 and TFAM genes from blastocysts was evaluated. The overall COCs recovery rate was 70.9 % (190/268). The maturation (57.8 vs 71.1), cleavage (45.2 vs 62.2) and blastocyst (30.1 vs 45.9) rates did not differ (P > 0.05) between RB and CR females, respectively. Similarly, no significant difference (P > 0.05) was observed for the expression of studied genes in both RB and CR females. In conclusion, oocytes obtained from RB were as developmentally competent as those collected from CR females, with similar energy metabolism and in vitro development capacity. Thus, the low fertility rate of repeat breeder buffaloes, when compared to normal cyclic females, must be due to subsequent events to the blastocyst stage.


Asunto(s)
Búfalos , Clima Tropical , Femenino , Animales , Búfalos/genética , Fertilización In Vitro/veterinaria , Oocitos/fisiología , Blastocisto/fisiología , Expresión Génica , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Desarrollo Embrionario/fisiología
9.
Biol Reprod ; 110(4): 672-683, 2024 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-38263524

RESUMEN

Chemically defined oocyte maturation media supplemented with FGF2, LIF, and IGF-1 (FLI medium) enabled significantly improved oocyte quality in multiple farm animals, yet the molecular mechanisms behind such benefits were poorly defined. Here, we first demonstrated that FLI medium enhanced mouse oocyte quality assessed by blastocyst formation after in vitro fertilization and implantation and fetal development after embryo transfer. We then analyzed the glucose concentrations in the spent media; reactive oxygen species concentrations; mitochondrial membrane potential; spindle morphology in oocytes; and the abundance of transcripts of endothelial growth factor-like factors, cumulus expansion factors, and glucose metabolism-related genes in cumulus cells. We found that FLI medium enabled increased glucose metabolism through glycolysis, pentose phosphate pathway, and hexosamine biosynthetic pathway, as well as more active endothelial growth factor-like factor expressions in cumulus cells, resulting in improved cumulus cell expansion, decreased spindle abnormality, and overall improvement in oocyte quality. In addition, the activities of MAPK1/3, PI3K/AKT, JAK/STAT3, and mTOR signaling pathways in cumulus cells were assessed by the phosphorylation of MAPK1/3, AKT, STAT3, and mTOR downstream target RPS6KB1. We demonstrated that FLI medium promoted activations of all these signaling pathways at multiple different time points during in vitro maturation.


Asunto(s)
Factor 2 de Crecimiento de Fibroblastos , Técnicas de Maduración In Vitro de los Oocitos , Animales , Ratones , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Factores de Crecimiento Endotelial/análisis , Factores de Crecimiento Endotelial/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Oocitos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Suplementos Dietéticos , Glucosa/farmacología , Glucosa/metabolismo , Células del Cúmulo/metabolismo
10.
Theriogenology ; 217: 37-50, 2024 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-38244353

RESUMEN

Heat stress (HS) is a stressor that negatively affect female reproduction. Specially, oocytes are very sensitive to HS. It has been demonstrated that some active compounds can protect oocyte from HS. We previously found that Mogroside V (MV), extracted from Siraitia grosvenorii (Luo Han Guo), can protect oocyte from many kinds of stresses. However, how MV alleviates HS-induced disruption of oocyte maturation remains unknown. In this study, we treated the HS-induced porcine oocytes with MV to examine their maturation and quality. Our findings demonstrate that MV can effectively alleviate HS-induced porcine oocyte abnormal cumulus cell expansion, decrease of first polar body extrusion rate, spindle assembly and chromosome separation abnormalities, indicating MV attenuates oocyte mature defects. We further observed that MV can effectively alleviate HS-induced cortical granule distribution abnormality and decrease of blastocyst formation rate after parthenogenesis activation. In addition, MV treatment reversed mitochondrial dysfunction and lipid droplet content decrease, reduced reactive oxygen species levels, early apoptosis and DNA damage in porcine oocytes after HS. Collectively, this study suggests that MV can effectively protect porcine oocytes from HS.


Asunto(s)
Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Triterpenos , Porcinos , Femenino , Animales , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oogénesis , Especies Reactivas de Oxígeno/farmacología , Respuesta al Choque Térmico
11.
Reproduction ; 167(4)2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38271822

RESUMEN

In brief: Pyruvate metabolism is one of the main metabolic pathways during oocyte maturation. This study demonstrates that pyruvate metabolism also regulates the epigenetic and molecular maturation in bovine oocytes. Abstract: Pyruvate, the final product of glycolysis, undergoes conversion into acetyl-CoA within the mitochondria of oocytes, serving as a primary fuel source for the tricarboxylic acid (TCA) cycle. The citrate generated in the TCA cycle can be transported to the cytoplasm and converted back into acetyl-CoA. This acetyl-CoA can either fuel lipid synthesis or act as a substrate for histone acetylation. This study aimed to investigate how pyruvate metabolism influences lysine 9 histone 3 acetylation (H3K9ac) dynamics and RNA transcription in bovine oocytes during in vitro maturation (IVM). Bovine cumulus-oocyte complexes were cultured in vitro for 24 h, considering three experimental groups: Control (IVM medium only), DCA (IVM supplemented with sodium dichloroacetate, a stimulant of pyruvate oxidation into acetyl-CoA), or IA (IVM supplemented with sodium iodoacetate, a glycolysis inhibitor). The results revealed significant alterations in oocyte metabolism in both treatments, promoting the utilization of lipids as an energy source. These changes during IVM affected the dynamics of H3K9ac, subsequently influencing the oocyte's transcriptional activity. In the DCA and IA groups, a total of 148 and 356 differentially expressed genes were identified, respectively, compared to the control group. These findings suggest that modifications in pyruvate metabolism trigger the activation of metabolic pathways, particularly lipid metabolism, changing acetyl-CoA availability and H3K9ac levels, ultimately impacting the mRNA content of in vitro matured bovine oocytes.


Asunto(s)
Histonas , Técnicas de Maduración In Vitro de los Oocitos , Animales , Bovinos , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Acetilcoenzima A/metabolismo , Histonas/metabolismo , Oocitos/metabolismo , Ácido Pirúvico/farmacología , Ácido Pirúvico/metabolismo , Epigénesis Genética , Células del Cúmulo
12.
Theriogenology ; 218: 16-25, 2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38290231

RESUMEN

Numerous growth factors contribute to oocyte maturation and embryonic development in vivo; however, only a few are understood. One such factor is epigen, a new member of the epidermal growth factor (EGF) family that is secreted by the granulosa cells of immature oocytes. We hypothesized that epigen may play a role in oocyte maturation, specifically in the nuclear and cytoplasmic aspects. This study aimed to investigate the effects of epigen on porcine oocyte maturation and embryo development in vitro. In this study, three different concentrations of epigen (3, 6, and 30 ng/mL) were added to tissue culture medium-199 (TCM-199) during in vitro maturation of porcine oocytes. A control group that did not receive epigen supplementation was also included. Mature porcine oocytes were fertilized, and the resulting zygotes were cultured until day 7. The levels of intracellular glutathione (GSH) and reactive oxygen species (ROS) were measured in the in vitro matured oocytes. At the same time, the expression patterns of genes related to apoptosis were detected in day 7 blastocysts (BLs) using real-time quantitative PCR Apoptosis was detected by annexin-V assays in mature oocytes. Data were analyzed using ANOVA and Duncan's test on SPSS, and results are presented as mean ± SEM. The group that received 6 ng/mL epigen had a significantly lower rate of germinal vesicle breakdown (GVBD) than the control group without affecting the nuclear maturation among the experimental groups. Among the treatment groups, the 6 ng/mL epigen group showed significantly higher levels of intracellular GSH and lower ROS production. Supplementation with 6 ng/mL epigen significantly improved blastocyst (BL) formation rates compared to those in the control and 3 ng/mL groups. Additionally, the blastocyst expansion rate was significantly higher with epigen supplementation (6 ng/mL). In the fertilization experiment, the group supplemented with 6 ng/mL epigen exhibited significantly higher levels of monospermy and fertilization efficiency and lower levels of polyspermy than the control group. This study indicated that adding epigen at a concentration of 6 ng/mL can significantly enhance the developmental potential of porcine oocytes fertilized in vitro. Specifically, the study found that epigen improves cytoplasmic maturation, which helps prevent polyspermy and emulates monospermic penetration.


Asunto(s)
Fertilización In Vitro , Fertilización , Embarazo , Femenino , Porcinos , Animales , Fertilización In Vitro/veterinaria , Fertilización In Vitro/métodos , Especies Reactivas de Oxígeno/farmacología , Epigen , Desarrollo Embrionario , Oocitos , Blastocisto , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos
13.
Theriogenology ; 218: 8-15, 2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38290232

RESUMEN

To investigate the effects of limonin (Lim) on oxidative stress and early apoptosis in bovine oocytes during in vitro maturation (IVM), different concentrations of Lim (0, 10, 20, 50 µmol/L) were added to bovine IVM medium. Oocyte maturation rates and development 24 h after in vitro fertilization (IVF) were examined to determine the optimal Lim concentration. The optimal Lim concentration was added to the IVM medium, and 0 µmol/L Lim was used as the control. Immunofluorescence staining was used to detect the abnormal rate of spindle assembly, reactive oxygen species (ROS), glutathione (GSH), mitochondrial membrane potential (MMP) levels, mitochondrial distribution, and the fluorescence intensity of cathepsin B (CB)-active LC3 protein. RT‒qPCR was used to detect the mRNA expression levels of antioxidant-, apoptosis- and autophagy-related genes in oocytes. The total number of blastocysts and the proportion of apoptotic cells among blastocysts were detected. The results showed that the PBI ejection rate, cleavage rate and blastocyst rate of bovine oocytes in the 20 µmol/L Lim group were significantly higher than those in the control group (P < 0.05). Compared with those in the control group, ROS levels, abnormal mitochondrial distribution, the proportion of abnormal spindle assembly, CB activity and LC3 protein fluorescence intensity of oocytes in the 20 µmol/L Lim group were significantly decreased (P < 0.05), and GSH and MMP levels were significantly increased (P < 0.05). The expression of antioxidant genes (Prdx3, Prdx6, Sirt1) and antiapoptotic genes (Bcl-xl, Survivin) were significantly upregulated (P < 0.05), and the expression levels of proapoptotic genes (Caspase-4, BAX) and autophagy-related genes (LC3) were significantly downregulated (P < 0.05). The total number of cells among in vitro fertilized embryos was significantly increased (P < 0.05), and the apoptosis rate of blastocysts was significantly decreased (P < 0.05). Here, we show that Lim exerts positive effects on bovine oocyte IVM by regulating REDOX homeostasis, reducing spindle damage and enhancing mitochondrial function during IVM, thereby inhibiting oocyte apoptosis and autophagy.


Asunto(s)
Antioxidantes , Limoninas , Animales , Bovinos , Antioxidantes/farmacología , Especies Reactivas de Oxígeno/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Limoninas/metabolismo , Limoninas/farmacología , Oocitos/fisiología , Estrés Oxidativo , Glutatión/metabolismo , Blastocisto/fisiología , Apoptosis , Desarrollo Embrionario
14.
Anim Reprod Sci ; 260: 107382, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38035499

RESUMEN

Mito-Q is a well-known mitochondria-specific superoxide scavenger. To our knowledge, the effect of Mito-Q on buffalo oocyte maturation and developmental competency of cloned embryos has not been examined. To investigate the effects of Mito-Q on the in vitro maturation (IVM) of buffalo oocytes and the developmental competence of cloned embryos, different concentration of Mito-Q were supplemented with IVM (0, 0.1, 0.5, 1, 2 µM) and in vitro culture (IVC) medium (0, 0.1 µM). Supplementation of IVM medium with 0.1 µM Mito-Q significantly (P ≤ 0.05) increased the cumulus expansion, nuclear maturation, mitochondrial membrane potential (MMP) and antioxidants genes (GPX1 and SOD2) expression and effectively reduced ROS production leading to a significant improvement in the maturation rate of buffalo oocytes. Further, the supplementation of 0.1 µM Mito-Q in IVC medium promotes the cleavage and blastocyst rate significantly over the control. Mito-Q supplementation improves (P ≤ 0.05) MMP, antioxidant gene (GPX1) expression and reduced the ROS level and apoptosis related genes (caspase 9) expression in cloned blastocysts. In conclusion, the present study demonstrated that the supplementation of 0.1 µM Mito-Q in IVM and IVC media exerts a protective role against oxidative stress by reducing ROS production and improving MMP, fostering improved maturation of buffalo oocytes and enhanced developmental competence of cloned embryos. These findings contribute valuable insights into the optimization of assisted reproductive technologies protocols for buffalo breeding and potentially offer novel strategies to enhance reproductive outcomes in livestock species.


Asunto(s)
Bison , Búfalos , Animales , Especies Reactivas de Oxígeno/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Oocitos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Blastocisto , Suplementos Dietéticos , Desarrollo Embrionario
15.
J Therm Biol ; 119: 103759, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38035528

RESUMEN

Heat stress is a significant factor affecting the fertility of dairy cattle due to the generation of free radicals. In assisted reproductive techniques, the inclusion of protective antioxidants becomes crucial to mitigate potential cellular damage. This study aimed to explore the impact of supplementing vitamins E, C, and coenzyme Q10 into the oocyte culture medium, with the goal of ameliorating the adverse effects of heat stress on oocyte maturation and embryo development in dairy cattle. A group of fifty Holstein dairy cows were synchronized, and their oocytes were harvested using the ovum pick-up method. High-quality oocytes were subjected to in vitro maturation (IVM) and in vitro fertilization (IVF) procedures, utilizing a culture medium containing, no supplements (Group 1), 100 µM of vitamins E (Group 2) and C (Group 3), along with 50 µM of coenzyme Q10 (Group 4). The ensuing zygotes were cultured, and the ensuing embryos were evaluated for blastocyst formation by the seventh day. An analysis of the blastocysts' inner cell mass (ICM) and trophectoderm (TE) cells was also conducted. The findings revealed that the group receiving supplementation of vitamin E and coenzyme Q10 exhibited significantly higher maturation and cleavage rates in comparison to both the control and the vitamin C groups. Furthermore, the count of ICM, TE, and blastocyst cells was notably elevated in the vitamin E supplemented group when compared to the control group. In summary, the effectiveness of vitamin E in enhancing IVM, IVF, and embryo development under conditions of heat stress surpassed that of vitamin C and coenzyme Q10.


Asunto(s)
Técnicas de Maduración In Vitro de los Oocitos , Ubiquinona/análogos & derivados , Vitamina E , Animales , Femenino , Bovinos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Vitamina E/farmacología , Oocitos , Desarrollo Embrionario , Suplementos Dietéticos , Vitaminas/farmacología , Ácido Ascórbico/farmacología , Respuesta al Choque Térmico
16.
Reprod Domest Anim ; 59(1): e14503, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37942895

RESUMEN

This study evaluated the effect of supplementing IVM media with γ-oryzanol (ORY), a nutraceutical derived from rice bran oil, on the development of bovine oocytes and hindering the compromising effect of redox imbalance. An in vitro model of the bovine cumulus-oocyte complex was used for the evaluation of nuclear maturation and development. Antioxidant activity was investigated by assessing the level of ROS (Reactive Oxygen Species) and GSH (glutathione) in oocytes and quantitative changes in gene expression in matured oocytes and their respective cumulus cells. ORY supplementation increased the proportion of MII oocytes, cleaved embryos, and total blastocysts (p < .05) and was linked to higher and lower levels of intracellular GSH and ROS, respectively (p < .05). The treated oocytes and their respective cumulus-granulosa cells showed a modulation in the expression of genes related to apoptosis (downregulation of BAX and CHOP) and oxidative stress (upregulation of NRF2, CAT, and SOD). Also, relative upregulation of OCT-4 and IGF2R in treated oocytes was concomitant with higher subsequent development in terms of cleavage and total blastocyst rates (p < .05). Based on our findings, it appears that ORY supplementation can improve the nuclear maturation and development of bovine oocytes into blastocysts and augment their enzymatic and non-enzymatic antioxidant systems, maintaining the Redox balance and high enzymatic activity against ROS generation.


Asunto(s)
Antioxidantes , Técnicas de Maduración In Vitro de los Oocitos , Fenilpropionatos , Femenino , Animales , Bovinos , Antioxidantes/farmacología , Antioxidantes/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos , Oxidación-Reducción , Glutatión/metabolismo , Blastocisto/metabolismo , Suplementos Dietéticos , Desarrollo Embrionario
17.
Theriogenology ; 215: 187-194, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38086312

RESUMEN

In postpartum dairy cows, lipopolysaccharide (LPS) derived from gram-negative bacteria causes uterine or mammary inflammation, resulting in low fertility. The present study aimed to investigate the effect of LPS on the in vitro growth (IVG), steroidogenesis, and maturation of oocyte-cumulus-granulosa cell complexes (OCGCs) derived from bovine early antral follicles. OCGCs were isolated from bovine early antral follicles (0.5-1 mm in diameter) and cultured in vitro for 12 days using media containing 0 (control), 0.01, or 1 µg/mL of LPS. The viability, cavity formation, and oocyte diameter of the OCGCs, as well as the concentrations of estradiol (E2) and progesterone (P4) in the IVG culture media, were determined. After IVG culture, oocytes collected from viable OCGCs were matured in vitro (IVM) in a medium without LPS. The nuclear maturation rate and the mitochondrial membrane potential of oocytes were determined. Bovine oocytes and cumulus-granulosa complexes derived from early antral follicles expressed genes encoding LPS receptor complex, such as toll-like receptor 4 (TLR4). Immunohistochemistry analysis further localized TLR4 expression predominantly in follicular granulosa and theca cells of early antral follicles. The viability of OCGCs and cavity formation in OCGCs were lower in the 0.01 and 1 µg/mL LPS groups than in the control group. No significant difference in oocyte diameter was observed between the treatment groups throughout the culture period. Moreover, E2 production was suppressed in the 0.01 and 1 µg/mL LPS groups from Days 4-8, whereas P4 production increased in the 1 µg/mL LPS group from Days 0-8. The nuclear maturation rate after IVM was lower in the 0.01 and 1 µg/mL LPS groups than in the control group. The mitochondrial membrane potential of post-IVM oocytes was lower in the 0.01 and 1 µg/mL LPS groups than in the control group. Taken together, these results indicate that LPS inhibited the growth and steroidogenesis of OCGCs and the meiosis and mitochondrial function of oocytes derived from early antral follicles. This study suggests that the detrimental effects of LPS on developing oocytes may contribute to long-term decreased fertility in postpartum dairy cows.


Asunto(s)
Lipopolisacáridos , Receptor Toll-Like 4 , Femenino , Bovinos , Animales , Lipopolisacáridos/farmacología , Receptor Toll-Like 4/metabolismo , Oocitos/fisiología , Células del Cúmulo/metabolismo , Meiosis , Técnicas de Maduración In Vitro de los Oocitos/veterinaria
18.
Biol Trace Elem Res ; 202(1): 161-174, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-37127784

RESUMEN

Selenium (Se), an essential trace element, plays an important role in the antioxidative defense mechanism, and it has been proven to improve fertility and reproductive efficiency in dairy cattle. The present study evaluated the potential protective action of Se supplement of in vitro maturation (IVM) media on the maturation and subsequent development of bovine cumulus-oocyte complexes (COCs) exposed to heat stress (HS). The treatment with Se improved the viability of cumulus cells (CCs) and oocytes (P < 0.05). The proportion of oocytes reached metaphase II (MII) and those arrested at metaphase I (MI) was greater and lower in treatment than control respectively (P < 0.05). Supplementation with Se increased the percentage of cleaved embryos, total blastocysts, and blastocyst/cleavage ratio (P < 0.05). Moreover, the upregulation of CCND1, SEPP1, GPX-4, SOD, CAT, and downregulation of GRP78, CHOP, and BAX in both Se-treated CCs and oocytes were recorded. The upregulation of NRF2 was detected in Se-treated CCs other than in oocytes, which showed upregulation of IGF2R and SOX-2 as the markers of quality as well. Se supplement in IVM media improved the viability, maturation, and the level of transcripts related to antioxidant defense and quality of heat-treated oocytes, which coincided with greater subsequent development outcomes. Se ameliorated the viability of CCs along with upregulation of antioxidative candidate gene expression and downregulation of apoptosis-related ones to support their protective role on restoring the quality of oocytes against compromising effects of HS.


Asunto(s)
Técnicas de Maduración In Vitro de los Oocitos , Selenito de Sodio , Bovinos , Animales , Femenino , Selenito de Sodio/farmacología , Selenito de Sodio/metabolismo , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Oocitos , Respuesta al Choque Térmico , Células del Cúmulo/fisiología
19.
Reproduction ; 167(2)2024 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-38038651

RESUMEN

In brief: Epigenetic programming is a crucial process during early embryo development that can have a significant impact on the results of assisted reproductive technology (ART) and offspring health. Here we show evidence using a bovine in vitro experiment that embryo epigenetic programing is dependent on oocyte mitochondrial bioenergetic activity during maturation. Abstract: This study investigated if oocyte and early embryo epigenetic programming are dependent on oocyte mitochondrial ATP production. A bovine in vitro experiment was performed in which oocyte mitochondrial ATP production was reduced using 5 nmol/L oligomycin A (OM; ATP synthase inhibitor) during in vitro maturation (IVM) compared to control (CONT). OM exposure significantly reduced mitochondrial ATP production rate in MII oocytes (34.6% reduction, P = 0.018) and significantly decreased embryo cleavage rate at 48 h post insemination (7.6% reduction, P = 0.031). Compared to CONT, global DNA methylation (5mC) levels were decreased in OM-exposed MII oocytes (9.8% reduction, P = 0.019) while global histone methylation (H3K9me2) was increased (9.4% increase, P = 0.024). In zygotes, OM exposure during IVM increased 5mC (22.3% increase, P < 0.001) and histone acetylation (H3K9ac, 17.3% increase, P = 0.023) levels, while H3K9me2 levels were not affected. In morulae, 5mC levels were increased (10.3% increase, P = 0.041) after OM exposure compared to CONT, while there was no significant difference in H3K9ac and H3K9me2 levels. These epigenetic alterations were not associated with any persistent effects on embryo mitochondrial ATP production rate or mitochondrial membrane potential (assessed at the four-cell stage). Also, epigenetic regulatory genes were not differentially expressed in OM-exposed zygotes or morulae. Finally, apoptotic cell index in blastocysts was increased after OM exposure during oocyte maturation (41.1% increase, P < 0.001). We conclude that oocyte and early embryo epigenetic programming are dependent on mitochondrial ATP production during IVM.


Asunto(s)
Histonas , Técnicas de Maduración In Vitro de los Oocitos , Animales , Bovinos , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Epigenoma , Oligomicinas/farmacología , Oocitos , Desarrollo Embrionario , Adenosina Trifosfato
20.
Theriogenology ; 215: 78-85, 2024 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-38016304

RESUMEN

During in vitro maturation (IVM) cumulus-oocyte complexes (COCs) are exposed to conditions that can trigger oxidative stress, thus, reducing oocyte maturation and viability. Aiming to mitigate these detrimental conditions, the effects of IVM medium supplementation with anethole have been tested. Anethole, also known as trans-anethole (1-methoxy-4 [1-propenyl]-benzene), is a naturally occurring phenylpropanoid with various pharmacological properties, including antioxidant effects. However, no study has examined anethole effect on goat COCs during IVM. Thus, the aim of this study was to evaluate the effects of different anethole concentrations on oocyte maturation, oxidative stress, and in vitro development of caprine embryos after parthenogenetic activation. Goat COCs were selected and randomly distributed into the following treatments: TCM-199+ medium (control), or TCM-199+ medium supplemented with 30 µg/mL (AN30); 300 µg/mL (AN300) or 2000 µg/mL (AN2000) of anethole. After IVM, part of the COCs was chosen for oocyte viability and chromatin configuration, intracellular reactive oxygen species levels, and mitochondrial membrane potential assessment. Another part of COCs was parthenogenetically activated, and presumptive zygotes were cultured for 7 days. Results demonstrated that anethole at 30 µg/mL increased oocyte maturation and cleavage rates when compared to the other treatments (P < 0.05), as well as oocyte viability and in vitro embryo production when compared to the control treatment (P < 0.05). Additionally, treatment with anethole at 2000 µg/mL decreased oocyte nuclear maturation and cleavage rates when compared to other treatments (P < 0.05) and embryo production if compared to control and AN30 treatments (P < 0.05). Moreover, anethole at 2000 µg/mL increased mitochondrial membrane potential when compared to the other treatments (P < 0.05). In conclusion, anethole exerts a concentration-dependent effect during goat COCs IVM. For a more desirable outcome of oocyte viability and maturation, and in vitro embryo production, the use of anethole at 30 µg/mL is recommended.


Asunto(s)
Cabras , Técnicas de Maduración In Vitro de los Oocitos , Animales , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Técnicas de Maduración In Vitro de los Oocitos/métodos , Cabras/fisiología , Oocitos/fisiología , Suplementos Dietéticos , Células del Cúmulo
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