Leptin has concentration and stage-dependent effects on embryonic development in vitro

There is accumulating evidence that leptin may be directly involved in pre-implantation embryonic development, however, it is unclear whether there is a concentration and stage-dependent regulatory pattern. In this study, the addition of 10 ng/ml human recombinant leptin to the culture medium significantly increased the percentage of two-cell mouse embryos that developed into blastocysts and hatched blastocysts, whereas in the presence of 100 ng/ml leptin, the development rate was significantly inhibited. The total cell numbers in the hatched blastocysts were significantly higher in the presence of 10 ng/ml leptin compared with controls and higher concentrations. The differential sensitivity to leptin was found to vary among embryos at different stages of development. Supplementation of leptin (10 ng/ml) to culture medium at two- to eight-cell stages resulted in a consistent stimulatory effect on embryo development. Most interestingly, the inhibitory effect of high leptin concentration (100 ng/ml) on embryo development was diminished when it was added to the culture medium at the eight-cell stage of development. The concentration-dependent regulation pattern was confirmed using sheep embryos, under similar conditions although sheep embryos appeared to be more sensitive in responding to leptin. Having established the effect of exogenous leptin on embryo development, the expression pattern of leptin and its receptors were also investigated. Leptin mRNA was not detected in mouse two-, four-, eight-cell and blastocyst stage embryos, whereas three isoforms of leptin receptor (Ob-Ra, Ob-Rb and Ob-Re) were identified in these cells, indicating that leptin is likely to modulate embryo development via a paracrine signalling system.

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146 Quercetin supplementation during boar semen thawing and incubation improves the in vitro production of pig embryos

Reproduction Fertility and Development ◽

10.1071/rdv31n1ab146 ◽

2019 ◽

Vol 31 (1) ◽

pp. 198

Author(s):

E. Hicks ◽

E. Winn ◽

B. Whitaker

Keyword(s):

Oxidative Stress ◽

Embryonic Development ◽

Developmental Stages ◽

Membrane Damage ◽

In Vitro Production ◽

Cell Stage ◽

Stage Of Development ◽

Boar Semen ◽

Morphological Deformities

Elevated levels of reactive oxygen species in the in vitro environment cause oxidative stress, which leads to membrane damage, decreased fertility, and morphological deformities of spermatozoa. Antioxidants, such as quercetin (a polyphenol flavonoid), are often supplemented to reduce the effects of oxidative stress on spermatozoa. Supplementing frozen-thawed boar semen with quercetin improves sperm forward progressive motility, viability and lipid peroxidation up to 10h after thawing. However, the effects of fertilizing with quercetin-supplemented sperm are unknown. Therefore, the objective of this study was to determine the effects of supplementing quercetin (0.25, 0.50, 0.75mM) during the thawing and incubation of frozen-thawed boar semen on oocyte fertilization characteristics (n=400) and subsequent embryonic development (n=1340) at 48 and 144h for cleavage and blastocyst formation, respectively. Oocytes from aspired aspirated mature follicles (3-6mm diameter) were obtained from a local abattoir and matured in medium 199 for 40 to 44h at 38.5°C in an atmosphere of 5% CO2. Fertilization was performed using pooled frozen-thawed semen from 3 different boars, and co-incubation of the sperm (2×105 sperm mL−1) and oocytes (30 oocytes/well) lasted for 6 to 8h at 38.5°C in an atmosphere of 5% CO2. Data were analysed using ANOVA with the main effects including treatment, well and replicate. Chi-squared analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no differences in penetration rates and male pronuclear formation between treatment groups; however, supplementation of 0.25 (18.18±10.63%), 0.50 (20.93±9.89%) and 0.75mM (18.07±12.02%) quercetin significantly decreased (P<0.05) polyspermic penetration rates compared with no supplementation (40.00±11.34%). Embryos produced from frozen-thawed boar sperm supplemented with 0.25 and 0.50mM quercetin had a significantly higher percentage (P<0.05) of embryos reaching the 2-cell stage of development by 48h after IVF (75.00±7.89%, 68.75±2.23%, respectively) compared with 0.75mM quercetin supplementation (64.62±3.88%) and no supplementation (62.97±4.11%). Supplementation of 0.25 (44.12±6.23%), 0.50 (43.75±7.02%) and 0.75mM (43.08±2.98%) quercetin to the sperm significantly increased (P<0.05) the percentage of embryos reaching the blastocyst stage of development by 144h after IVF compared with no supplementation (28.27±8.07%). These results indicate that supplementing frozen-thawed boar semen with quercetin decreases the incidence of polyspermic penetration and improves early embryonic development in pigs.

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Effect of RU486 on different stages of mouse preimplantation embryos in vitro

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y90-220 ◽

1990 ◽

Vol 68 (11) ◽

pp. 1457-1460 ◽

Cited By ~ 6

Author(s):

Subhash C. Juneja ◽

Melvin G. Dodson

Keyword(s):

Embryo Development ◽

Culture Medium ◽

Inhibitory Effect ◽

Blastocyst Stage ◽

Preimplantation Embryos ◽

In Vitro Development ◽

Stage Embryo ◽

Morula Stage ◽

Vitro Development

17β-Hydroxy-11β-(4-dimethylaminophenyl)-17α-(1-propynyl)estra-4,9-dien-3-one (RU486) inhibited the in vitro development of different stages of mouse preimplantation embryos under study. Two-celled embryos, morulae, and early blastocysts were obtained from B6D2F1 mice. The embryos were grown in Ham F-10 nutrient mixture (with glutamine) supplemented with sodium bicarbonate (2.1 g/L), calcium lactate (282 mg/L), and bovine serum albumin (fraction V, 3 mg/mL) at 37 °C in a humidified incubator supplied with 5% CO2 in air. RU486 was added to the culture medium at concentrations of 1, 5, 10, and 20 μg/mL. Culture medium with 0.05% ethanol served as the control. In vitro growth of embryos was assessed by the following criteria: (i) two-celled stage embryo development to blastocyst stage after 72 h, (ii) morula stage grown to blastocyst stage after 24 h, and (iii) early blastocyst stage development to hatching blastocyst after 12 h, in culture. RU486 inhibited the in vitro development of two-celled embryos, morulae, and early blastocysts at concentrations of 5, 10, and 20 μg/mL culture medium (p < 0.001). The inhibitory effect of RU486 at these concentrations on the development of all the stages of embryos under study was irreversible. However, RU486 did not affect embryo development at 1 μg/mL culture medium. The study indicates the direct adverse effect of RU486 at 5 μg/mL and higher concentrations in culture medium on the development of mouse preimplantation embryos in vitro, and it encourages its further investigation as a postcoital contraceptive in animal models and humans.Key words: RU486, mouse, preimplantation embryos, embryo culture, postcoital contraceptive.

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248 EMBRYO DEVELOPMENT IN MICRODROPS AND MICROCHANNELS: COMPARISON OF NCSU23 SEQUENTIAL AND NON-SEQUENTIAL CULTURE MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv17n2ab248 ◽

2005 ◽

Vol 17 (2) ◽

pp. 274

Author(s):

A.S. Lima ◽

C.E. Ferguson ◽

M.B. Wheeler

Keyword(s):

Embryo Development ◽

Culture Medium ◽

Culture Media ◽

Cumulus Cells ◽

Blastocyst Stage ◽

The Other ◽

Cell Stage ◽

Development Rates ◽

Hatching Rates

The in vitro culture systems used to produce pig embryos generally result in few embryos developing to the blastocyst stage. The use of pyruvate (pyr) and lactate (lac) during the culture of zygotes to the 8-cell stage followed by glucose (glu) supplementation replacing pyr and lac appears to be beneficial for embryo development in the pig. The aim of this study was to compare the embryo development rates from pig oocytes fertilized with and without cumulus cells in 100-μL microdrops (MD) and cultured in 100-μL MD or microchannels (MC), using NCSU23 containing 8 mg/mL of BSA and supplemented with (1) glu or (2) pyr/lac or (3) pyr/lac for the first three days and then with just glu for the remainder of culture period (pyr/lac-glu). Sow oocytes were matured in TCM199 supplemented with gonadotropins for the first 22 h, and for an additional 22 h without hormones. After 44 h of maturation, oocytes were placed in MD of modified tris-buffered medium to be fertilized using 3 × 105 sperm/mL. Oocytes were divided into two groups for fertilization: with and without cumulus cells. Following 6 h of fertilization, all inseminated oocytes were washed, divided into groups of 15, allotted to the three culture media treatment groups as described above, and incubated in either MD or MC. With the exception of one treatment there were no significant differences in development rates among embryos cultured in MD or MC, hence data were pooled from these two culture devices. Only oocytes fertilized without cumulus cells and cultured in pyr/lac in MC appeared to have lower rates of blastocyst formation (11.67%) than those cultured in MD (26.67%) in the same culture medium. When the six treatments were compared, oocytes fertilized with cumulus cells and cultured in glu had significantly higher (P < 0.05) blastocyst rates and hatching rates compared with the other treatments, with the exception of those fertilized without cumulus cells and cultured in pyr/lac-glu. There were no significant differences among other treatments in Day 7 blastocyst or in Day 9 hatching rates. In conclusion, both culture devices can be used to reach similar blastocyst rates with different treatments. In this experiment, the removal of cumulus cells before fertilization appeared to enhance embryo development in vitro when sequential media are used. On the other hand, the presence of cumulus cells before fertilization seems to enhance embryo development when non-sequential glu medium is used. Table 1. Embryo development rates on Day 9 for three different culture treatments

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BPA and BPS Affect the Expression of Anti-Mullerian Hormone (AMH) and Its Receptor During Bovine Oocyte Maturation and Early Embryo Development

10.21203/rs.3.rs-435109/v1 ◽

2021 ◽

Author(s):

Angela Christina Saleh ◽

Reem Sabry ◽

Gabriela Fabiana Mastromonaco ◽

Laura Alessandra Favetta

Keyword(s):

Embryonic Development ◽

Embryo Development ◽

Oocyte Maturation ◽

Quantitative Polymerase Chain Reaction ◽

Early Embryo ◽

Bisphenol S ◽

Cell Stage ◽

Vitro Fertilization ◽

Preimplantation Embryo Development

Abstract Background Exposure to endocrine-disrupting chemicals, such as Bisphenol A (BPA) and Bisphenol S (BPS), is widespread and has negative implications on embryonic development. Preliminary evidence revealed that in women undergoing IVF treatment, urinary BPA levels were associated with low serum anti-Mullerian hormone, however a definitive relationship between the two has not yet been characterized. Methods This study aimed to evaluate BPA and BPS effects on in vitro oocyte maturation and early preimplantation embryo development through i) analysis of anti-Mullerian hormone (AMH) and anti-Mullerian hormone receptor II (AMHRII), ii) investigation of developmental parameters, such as cleavage, blastocyst rates and developmental arrest, iii) detection of apoptosis and iv) assessment of possible sex ratio skew. An in vitro bovine model was used as a translational model for human early embryonic development. We first assessed AMH and AMHRII levels after bisphenol exposure during oocyte maturation. Zygotes were also analyzed during cleavage and blastocysts stages. Techniques used include in vitro fertilization, quantitative polymerase chain reaction (qPCR), western blotting, TUNEL and immunofluorescence. Results Our findings show that BPA significantly decreased cleavage (p < 0.001), blastocyst (p < 0.005) and overall developmental rates as well as significantly increased embryonic arrest at the 2–4 cell stage (p < 0.05). Additionally, both BPA and BPS significantly increased DNA fragmentation in 2–4 cells, 8–16 cells and blastocyst embryos (p < 0.05). Furthermore, BPA and BPS alter AMH and AMHRII at the mRNA and protein level in both oocytes and blastocysts. BPA, but not BPS, also significantly skews sex ratios towards female blastocysts (p < 0.05) Conclusion This study shows that BPA affects AMH and AMHRII expression during oocyte maturation and that BPS exerts its effects to a greater extent after fertilization and therefore may not be a safer alternative to BPA. Our data lay the foundation for future functional studies, such as receptor kinetics, downstream effectors, and promoter activation/inhibition to prove a functional relationship between bisphenols and the AMH signalling system.

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189 EFFECT OF PROGESTERONE AND EPIDERMAL GROWTH FACTOR ON IN VITRO-PRODUCED EIGHT-CELL BOVINE EMBRYOS IN A SERUM-FREE CULTURE MEDIUM

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab189 ◽

2007 ◽

Vol 19 (1) ◽

pp. 211 ◽

Cited By ~ 9

Author(s):

B. Merlo ◽

E. Iacono ◽

G. Mari

Keyword(s):

Growth Factor ◽

Embryo Development ◽

Culture Medium ◽

Bovine Embryo ◽

Bovine Embryos ◽

Cell Stage ◽

Serum Free ◽

Epidermal Growth ◽

Blastocyst Rate

The role of progesterone (P4) and epidermal growth factor (EGF) in early bovine embryo development is still not clear. P4 has been administered at different times of embryo development, and a direct effect on IVF-derived bovine 8-cell embryos has been noted even if there was an interference due to the P4 vehicle (Ferguson et al. 2005 Reprod. Fertil. Dev. 17, 219 abst). EGF has been added to the culture medium from the presumptive zygote stage at different concentrations, resulting in improved blastocyst rates compared to that in control medium (Mtango et al. 2003 Theriogenology 59, 1393–1402; Sirisathien et al. 2003 Anim. Reprod. Sci. 77, 21–32), and gave results similar to those with 5% or 10% FCS (Palasz et al. 2000 Anim. Reprod. Sci. 58, 229–240). The objective if this experiment was to determine the effect of P4 and EGF on development of in vitro-produced bovine embryos when administered alone or in combination at the 8-cell stage in the absence of serum. In vitro-produced bovine 8-cell embryos were randomly allotted to treatments: (1) control, SOFaaBSA medium (BSA, 16 mg mL−1; n = 198); (2) P4, SOFaaBSA + P4 (15 ng mL−1 in ethanol; n = 198); (3) EGF, SOFaaBSA + EGF (25 ng mL−1; n = 200); (4) P4 + EGF, SOFaaBSA + P4 (15 ng mL−1 in ethanol) + EGF (25 ng mL−1; n = 201); and (5) FBS, SOFaaBSA + FBS (5%; n = 197). In order to minimize the toxic effect of ethanol, it was allowed to evaporate from the culture dish and then medium was added. All in vitro procedures were carried out at 38.5°C in a humidified atmosphere of 5% CO2 in air; presumptive zygotes were cultured in SOFaaBSA until 8-cell stage. Embryo development was evaluated on Day 6 and on Day 8 after IVF (Day 0), and rates calculated from 8-cell embryos. The study was done in 4 replicates and chi-square test was used for statistical analysis (Statistica for Windows; Stat Soft Inc., Tulsa, OK, USA); significance was assessed at P < 0.05. Results are reported in Table 1. No differences were found in the number of morulae between P4 and control, between P4 + EGF and FBS, and between P4 + EGF and EGF (P > 0.05), whereas the combination P4 + EGF was better than P4 alone (P < 0.05). Blastocyst rate was not different (P > 0.05) among EGF, P4 + EGF, and FBS groups. P4 achieved an higher (P < 0.05) blastocyst rate than control but it was lower (P < 0.05) than that of P4 + EGF or FBS. In conclusion, P4 alone improves embryo development from the 8-cell embryo to the blastocyst stage in a serum-free culture system, and EGF alone achieves a blastocyst rate not significantly different from that of FBS; furthermore, the combination of P4 and EGF can be considered the most suitable as an alternative to FBS because similar results were obtained in terms of both morulae and blastocysts. Table 1.Eight-cell bovine embryo development in SOFaaBSA medium in presence of P4, EGF, P4+EGF, or FBS

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156 OVIDUCTAL FLUID SUPPLEMENTATION DURING MATURATION AND FERTILIZATION IMPROVES IN VITRO EMBRYONIC DEVELOPMENT IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab156 ◽

2017 ◽

Vol 29 (1) ◽

pp. 187

Author(s):

A. Goldacker ◽

E. Winn ◽

J. Z. Current ◽

B. D. Whitaker

Keyword(s):

Embryonic Development ◽

Developmental Stages ◽

Blastocyst Stage ◽

Success Rates ◽

Blastocyst Development ◽

Cell Stage ◽

Stage Of Development ◽

Fluid Supplementation ◽

Chi Square Analysis

Oviducal fluid has a major role in the maturation of gametes and the process of fertilization. The objective of this study was to determine the effects of oviducal fluid supplementation in vitro, during oocyte maturation and IVF on fertilization characteristics and early embryonic development rates. Oocytes from aspired aspirated mature follicles (3–6 mm diameter) were obtained from a local abattoir. During the last 24 h of maturation, oocytes (n = 1303) were placed into maturation media supplemented either 1% (vol/vol) or 5% (vol/vol) thawed snap-frozen oviducal fluid. Fertilization was performed using pooled frozen-thawed semen from 3 different boars. During IVF, the fertilization medium was supplemented with 1% (vol/vol) or 5% (vol/vol) oviducal fluid. Fertilization characteristics were evaluated 12 h after IVF and rates of embryonic cleavage and blastocyst development were observed at 48 and 144 h after IVF, respectively. Data were analysed using ANOVA with the main effects including treatment, well, and replicate. Chi-square analysis was used to determine percentages of embryos reaching the different developmental stages for each treatment. There were no significant differences in the percentages of oocytes that reached metaphase II by the end of maturation or in sperm penetration rates after IVF. However, oocytes treated with 1% (vol/vol) oviducal fluid during the end of maturation and IVF (33.33 ± 2.61) and 5% (vol/vol) oviducal fluid during maturation (33.33 ± 2.66) or IVF (39.53 ± 3.78) had significantly less (P < 0.05) incidence of polyspermic penetrations and a significantly higher (P < 0.05) incidence of male pronuclear formation (87.50 ± 4.01; 86.67 ± 4.83; 86.05 ± 3.19, respectively) compared with no oviducal fluid supplementation. Oocytes supplemented with 5% (vol/vol) oviducal fluid during maturation and IVF had significantly lower (P < 0.05) incidences of polyspermic penetration (27.91 ± 2.50) and significantly higher (P < 0.05) percentages of embryos reaching the 2-cell stage (81.76 ± 3.72) and blastocyst stage of development (37.74 ± 1.09) by 48 and 144 h, respectively, compared with all other groups. The results of this study suggest that supplementing 5% (vol/vol) oviducal fluid during maturation and IVF improves the success rates of in vitro embryo development in pigs.

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Development of four-cell hamster embryos to the blastocyst stage in vitro and its regulation by components of the culture milieu

Reproduction Fertility and Development ◽

10.1071/rd9900001 ◽

1990 ◽

Vol 2 (1) ◽

pp. 1 ◽

Cited By ~ 11

Author(s):

H Monis ◽

BD Bavister

Keyword(s):

Amino Acids ◽

Co2 Concentration ◽

Inhibitory Effect ◽

Defined Medium ◽

Cell Number ◽

Blastocyst Stage ◽

Blastocyst Development ◽

Cell Stage ◽

Stage Of Development

Constituents of the culture milieu known to influence development of hamster 2-cell and 8-cell embryos were examined for effects on the 4-cell stage. Embryos were collected at the mid 4-cell stage (approx. 45-46 h after egg activation) from superovulated females and cultured for 24 h in a chemically defined medium (TLP-PVA). As with the 2-cell stage, inorganic phosphate (Pi) strongly inhibited development of 4-cell embryos, although some (14%) were able to reach the 8-cell stage or further in the presence of Pi. However, unlike 2-cell embryos, no significant inhibitory effect of glucose on development of 4-cell embryos was found. In the absence of glucose and Pi, development of 4-cell embryos was sensitive to amino acids in the medium: the mean cell number was increased using 21 amino acids compared with 4 amino acids, similarly to the 2-cell stage; however, late blastocyst development (blastocele formation) from 4-cell embryos was reduced using 21 compared with 4 amino acids, as with 8-cell embryos. Similarly to the 2-cell and 8-cell stages, raising the CO2 concentration from 5% to 10% in the gas atmosphere for culture increased the percentage of total blastocysts developing from the 4-cell stage, but did not affect the proportions of late-stage blastocysts. These data show that 4-cell-stage hamster embryos are somewhat similar to 2-cell embryos with respect to the regulation of development by constituents of the culture milieu, but, to some extent, the 4-cell embryo is a transitional stage of development.

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Reactive oxygen species in reproduction: harmful, essential or both?

Zygote ◽

10.1017/s0967199420000179 ◽

2020 ◽

Vol 28 (4) ◽

pp. 255-269

Author(s):

M. Jamil ◽

H. Debbarh ◽

S. Aboulmaouahib ◽

O. Aniq Filali ◽

K. Mounaji ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Embryonic Development ◽

Embryo Development ◽

Second Messengers ◽

Reactive Oxygen ◽

Cellular Tissue ◽

Oxygen Species ◽

Stage Of Development

SummaryThe process of embryonic development is crucial and radically influences preimplantation embryo competence. It involves oocyte maturation, fertilization, cell division and blastulation and is characterized by different key phases that have major influences on embryo quality. Each stage of the process of preimplantation embryonic development is led by important signalling pathways that include very many regulatory molecules, such as primary and secondary messengers. Many studies, both in vivo and in vitro, have shown the importance of the contribution of reactive oxygen species (ROS) as important second messengers in embryo development. ROS may originate from embryo metabolism and/or oocyte/embryo surroundings, and their effect on embryonic development is highly variable, depending on the needs of the embryo at each stage of development and on their environment (in vivo or under in vitro culture conditions). Other studies have also shown the deleterious effects of ROS in embryo development, when cellular tissue production overwhelms antioxidant production, leading to oxidative stress. This stress is known to be the cause of many cellular alterations, such as protein, lipid, and DNA damage. Considering that the same ROS level can have a deleterious effect on the fertilizing oocyte or embryo at certain stages, and a positive effect at another stage of the development process, further studies need to be carried out to determine the rate of ROS that benefits the embryo and from what rate it starts to be harmful, this measured at each key phase of embryonic development.

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BPA and BPS affect the expression of anti-Mullerian hormone (AMH) and its receptor during bovine oocyte maturation and early embryo development

Reproductive Biology and Endocrinology ◽

10.1186/s12958-021-00773-6 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Angela Christina Saleh ◽

Reem Sabry ◽

Gabriela Fabiana Mastromonaco ◽

Laura Alessandra Favetta

Keyword(s):

Embryonic Development ◽

Embryo Development ◽

Oocyte Maturation ◽

Quantitative Polymerase Chain Reaction ◽

Early Embryo ◽

Bisphenol S ◽

Cell Stage ◽

Vitro Fertilization ◽

Preimplantation Embryo Development

Abstract Background Exposure to endocrine-disrupting chemicals, such as Bisphenol A (BPA) and Bisphenol S (BPS), is widespread and has negative implications on embryonic development. Preliminary evidence revealed that in women undergoing IVF treatment, urinary BPA levels were associated with low serum anti-Mullerian hormone, however a definitive relationship between the two has not yet been characterized. Methods This study aimed to evaluate BPA and BPS effects on in vitro oocyte maturation and early preimplantation embryo development through i) analysis of anti-Mullerian hormone (AMH) and anti-Mullerian hormone receptor II (AMHRII), ii) investigation of developmental parameters, such as cleavage, blastocyst rates and developmental arrest, iii) detection of apoptosis and iv) assessment of possible sex ratio skew. An in vitro bovine model was used as a translational model for human early embryonic development. We first assessed AMH and AMHRII levels after bisphenol exposure during oocyte maturation. Zygotes were also analyzed during cleavage and blastocysts stages. Techniques used include in vitro fertilization, quantitative polymerase chain reaction (qPCR), western blotting, TUNEL and immunofluorescence. Results Our findings show that BPA significantly decreased cleavage (p < 0.001), blastocyst (p < 0.005) and overall developmental rates as well as significantly increased embryonic arrest at the 2–4 cell stage (p < 0.05). Additionally, both BPA and BPS significantly increased DNA fragmentation in 2–4 cells, 8–16 cells and blastocyst embryos (p < 0.05). Furthermore, BPA and BPS alter AMH and AMHRII at the mRNA and protein level in both oocytes and blastocysts. BPA, but not BPS, also significantly skews sex ratios towards female blastocysts (p < 0.05). Conclusion This study shows that BPA affects AMH and AMHRII expression during oocyte maturation and that BPS exerts its effects to a greater extent after fertilization and therefore may not be a safer alternative to BPA. Our data lay the foundation for future functional studies, such as receptor kinetics, downstream effectors, and promoter activation/inhibition to prove a functional relationship between bisphenols and the AMH signalling system.

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Nobiletin enhances the development and quality of bovine embryos in vitro during two key periods of embryonic genome activation

Scientific Reports ◽

10.1038/s41598-021-91158-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Karina Cañón-Beltrán ◽

Yulia N. Cajas ◽

Serafín Peréz-Cerezales ◽

Claudia L. V. Leal ◽

Ekaitz Agirregoitia ◽

...

Keyword(s):

Embryonic Development ◽

Signaling Pathway ◽

Akt Signaling ◽

Mitochondrial Activity ◽

Bovine Embryos ◽

Akt Signaling Pathway ◽

Cell Stage ◽

Development In Vitro

AbstractIn vitro culture can alter the development and quality of bovine embryos. Therefore, we aimed to evaluate whether nobiletin supplementation during EGA improves embryonic development and blastocyst quality and if it affects PI3K/AKT signaling pathway. In vitro zygotes were cultured in SOF + 5% FCS (Control) or supplemented with 5, 10 or 25 µM nobiletin (Nob5, Nob10, Nob25) or with 0.03% dimethyl-sulfoxide (CDMSO) during minor (2 to 8-cell stage; MNEGA) or major (8 to 16-cell stage; MJEGA) EGA phase. Blastocyst yield on Day 8 was higher in Nob5 (42.7 ± 1.0%) and Nob10 (44.4 ± 1.3%) for MNEGA phase and in Nob10 (61.0 ± 0.8%) for MJEGA phase compared to other groups. Mitochondrial activity was higher and lipid content was reduced in blastocysts produced with nobiletin, irrespective of EGA phase. The mRNA abundance of CDK2, H3-3B, H3-3A, GPX1, NFE2L2 and PPARα transcripts was increased in 8-cells, 16-cells and blastocysts from nobiletin groups. Immunofluorescence analysis revealed immunoreactive proteins for p-AKT forms (Thr308 and Ser473) in bovine blastocysts produced with nobiletin. In conclusion, nobiletin supplementation during EGA has a positive effect on preimplantation bovine embryonic development in vitro and corroborates on the quality improvement of the produced blastocysts which could be modulated by the activation of AKT signaling pathway.

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