Follicular environment as a predictive tool for embryo development and kinetics in cattle

2019 ◽  
Vol 31 (3) ◽  
pp. 451 ◽  
Author(s):  
Gláucia Pereira Alves ◽  
Fernanda Bertuccez Cordeiro ◽  
Camila Bruna de Lima ◽  
Kelly Annes ◽  
Érika Cristina dos Santos ◽  
...  
Keyword(s):  
Embryo Development ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Large Scale ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Fluid Composition ◽  
Predictive Tool ◽  
Transcription Pattern ◽  
Transcriptional Pattern

Follicular fluid composition and the transcription pattern of granulosa cells were analysed to better comprehend associations between embryo development and morphokinetics. Bovine follicles were punctured and their respective follicular fluid and granulosa cells were collected. Cumulus–oocyte complexes derived from these follicles were matured and fertilised invitro. Embryo morphology and kinetics were evaluated at 40h after insemination, when embryos were classified as fast (FCL, four or more cells), slow (SCL, 2–3 cells) or non-cleaved (NCL). Their development was followed until the blastocyst stage. Glucose, pyruvate, cholesterol and oestradiol were quantified in the follicular fluid and the transcription pattern of 96 target genes was evaluated in granulosa cells by large-scale quantitative reverse transcription polymerase chain reaction. Follicular fluid from the blastocyst group had increased levels of glucose, total cholesterol and pyruvate compared to the non-blastocyst group, whereas higher levels of oestradiol were observed in the follicular fluid of embryos and blastocysts with fast cleavage. The transcriptional pattern revealed altered metabolic pathways between groups, such as lipid metabolism, cellular stress and cell signalling. In conclusion, both follicular fluid and granulosa cells are associated with the possibility of identifying follicles that may generate embryos with high potential to properly develop to the blastocyst stage.

МОДЕЛИРОВАНИЕ СИСТЕМ ЭКСТРАКОРПОРАЛЬНОГО СОЗРЕВАНИЯ ООЦИТОВ КРУПНОГО РОГАТОГО СКОТА С ИСПОЛЬЗОВАНИЕМ СТРУКТУРНЫХ КОМПОНЕНТОВ ОВАРИАЛЬНЫХ ФОЛЛИКУЛОВ *

2019 ◽  
pp. 20-22
Author(s):  
T.I. KUZMINA ◽  
I.V. CHISTYAKOVA
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Bovine Serum ◽  
Egg Quality ◽  
Fetal Bovine Serum ◽  
Reproductive Technologies ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Cultural Systems ◽  
Fetal Bovine

Создание эффективной унифицированной системы дозревания донорских ооцитов обеспечит повышение результативности инновационных клеточных репродуктивных технологий. В исследовании проведен сравнительный мониторинг показателеймейотического созревания ооцитов коров, созревших в различных системах, дополненных структурными компонентами фолликулов (СКФ стенки фолликулов, клетки гранулезы, белки) и фолликулярной жидкостью,а также потенций к развитию из них доимплантационных эмбрионов. Анализу подверглись ооциты, прокультивированные в следующих системах:среда ТС199 с добавлением 10 фетальной бычьей сыворотки (ФБС), 50 мкг/мл эстрадиола, 10 мкг/мл лютеинизирующего гормона (ЛГ), 10 мкг/мл фолликулостимулирующего гормона (ФСГ) среда ТС199 с 10 эстральной сывороткой коров среда ТС199 с 50 жидкости из фолликулов диаметром 9 мм среда ТС199 с добавлением белков фолликулярной жидкости молекулярной массой 65 кДасреда ТС199 с 10 ФБС и 1106 клеток гранулезы среда ТС199 с 10 ФБС и тканью фолликула. В культуральные среды ко всем исследованным группам ооцитов добавляли антибиотики. Использование CКФ обеспечило значительное снижение доли ооцитов с дегенерированным хроматином, что способствовало увеличению уровня доимпланационных эмбрионов на стадии бластоцисты. Так, доля бластоцист, развившихся из ооцитов, созревших в среде со стенками фолликулов,составила43,5. В этой же группе выявлен минимальный уровень дегенерированных зародышей (6,45). Полученные данные предлагается использовать при моделировании систем дозревания ооцитов коров с целью повышения качества яйцеклеток.The creation of an effective unified maturation system of donor oocytes provides an increase in the efficiency of innovative cellular reproductive technologies. The comparative analysis of the meiotic maturation indicators of bovine oocytes, which were matured in different cultural systems modified by follicular structural components (FSC follicular walls, granulosa cells, proteins) and follicular fluid, as well as the potential for preimplantation embryonic development were evaluated in this study. Oocytes matured in following cultural systems: medium TC199 supplemented with 10 fetal bovine serum and 50 g/ml of estradiol, 10 g/ml of luteinizing hormone (LH), 10 g/ml of folliclestimulating hormone (FSH) medium TC199 with 10 estrous cow serum medium TC199 with 50 liquid from follicles with a diameter of 9 mm medium TC199 supplemented with the follicular fluid proteins with molecular weight 65 kDa medium TC199 with 10 fetal bovine serum and 1106 granulosa cells medium TC199 with the addition of 10 fetal bovine serum and follicle tissues were analyzed. Antibiotics were added to cultural media of all experimental groups of oocytes. The usage of FSC ensured the decrease in the proportion of oocytes with degenerated chromatin, which contribute the rise of the level of preimplantation embryos at the blastocyst stage. Thus, the proportion of blastocysts developed from oocytes matured in medium supplemented with follicular walls was 43.5. In the same experimental group, the number of degenerated embryos was 6.45. The obtained data are supposed to be used for modeling the cultural systems of cow oocytes in order to improve the egg quality.

Changes of follicular fluid composition during estrous cycle: The effects on oocyte maturation and embryo development in vitro

2019 ◽  
Author(s):  
A.A. Mohammed ◽  
T. Al-Shaheen ◽  
S. Al-Suwaiegh
Keyword(s):  
Embryo Development ◽  
Oocyte Maturation ◽  
Follicular Fluid ◽  
Extracellular Fluid ◽  
Cumulus Cells ◽  
Fluid Composition ◽  
Lh Surge ◽  
Antral Follicles ◽  
Positive Effects

Oocytes are bathed in extracellular fluid of the antral follicles, which is termed follicular fluid (FF). Follicular fluid is synthesized from secretions of theca, granulosa, and cumulus cells and from a transudate of blood plasma. Oocytes persist in meiotic arrest in antral follicles until luteinizing hormone (LH) surge or removal the oocytes from the ovarian follicles. This suggests that FF before LH surge might contain meiosis inhibiting factor(s). The microvasculatory bed of the follicular wall and the composition of FF undergo changes during follicular growth and development, which is important for oocyte maturation and subsequent embryo development. Therefore, it is expected that FF composition and components might change according to timing of FF aspiration from follicles. Hence, negative or positive effects could be expected when FF supplemented during oocyte maturation in vitro. Nutrition effects on microvasculatory bed of follicles and their sizes. Thus, the nutritional status of animals is a factor affected on oocyte maturation and embryo development. The present article reviews and discusses these effects.

scholarly journals Seasonal changes in bovine fertility: relation to developmental competence of oocytes, membrane properties and fatty acid composition of follicles

Reproduction ◽  
2001 ◽  
pp. 447-454 ◽  
Author(s):  
Y Zeron ◽  
A Ocheretny ◽  
O Kedar ◽  
A Borochov ◽  
D Sklan ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid Composition ◽  
Fatty Acid ◽  
Acid Composition ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Chemical Activation ◽  
Blastocyst Stage ◽  
Membrane Properties ◽  
Lipid Phase

Follicle dynamics and oocyte viability in Holstein primiparous and multiparous cows and the relationships between fertility and the biochemical and physical properties of oocyte membranes with season were examined. The conception rates of primiparous (n = 70 885) and multiparous (n = 143 490) cows differed, peaking in the winter and decreasing in the summer. The number of follicles 3-8 mm in diameter per ovary was higher in winter (19.6) compared with summer (12.0). However, in winter the percentage of ovaries with fewer than ten follicles per ovary was 16%, in contrast to 50% in summer. After aspiration of follicles, 7.5 oocytes per ovary were found in winter and 5.0 oocytes per ovary in summer. Cleavage to the two- to four-cell stage after chemical activation was greater in winter than in summer; this was enhanced at the morula stage and embryo development to the blastocyst stage was significantly higher in winter than in summer. Determination of the lipid phase transition in oocyte membranes revealed a shift of 6 degrees C between summer and winter. Fatty acid composition of phospholipids from follicular fluid, granulosa cells and oocytes indicated that there was a higher percentage of saturated fatty acids during the summer and that the percentages of mono-unsaturated and polyunsaturated fatty acids were higher in oocytes and granulosa cells during the winter. Oocytes and granulosa cells had similar fatty acid compositions, in contrast to follicular fluid. These results may explain the differences in the ability of oocytes to develop to the blastocyst stage at different seasons. Thus, temperature changes may lead to changes in membrane properties, which, in turn, can influence oocyte function and fertility.

scholarly journals Embryonic disc formation following post-hatching bovine embryo development in vitro

Reproduction ◽  
2020 ◽  
Vol 160 (4) ◽  
pp. 579-589 ◽  
Author(s):  
Priscila Ramos-Ibeas ◽  
Ismael Lamas-Toranzo ◽  
Álvaro Martínez-Moro ◽  
Celia de Frutos ◽  
Alejandra C Quiroga ◽  
...  
Keyword(s):  
Embryo Development ◽  
Blastocyst Stage ◽  
Embryo Morphology ◽  
Bovine Embryo ◽  
Pluripotency Marker ◽  
Embryonic Disc ◽  
Development In Vitro ◽  
Apoptosis And Necrosis ◽  
Disc Formation

Failures during conceptus elongation are a major cause of pregnancy losses in ungulates, exerting a relevant economic impact on farming. The developmental events occurring during this period are poorly understood, mainly because this process cannot be recapitulated in vitro. Previous studies have established an in vitro post-hatching development (PHD) system that supports bovine embryo development beyond the blastocyst stage, based on agarose gel tunnels and serum- and glucose-enriched medium. Unfortunately, under this system embryonic disc formation is not achieved and embryos show notorious signs of apoptosis and necrosis. The objective of this study has been to develop an in vitro system able to support embryonic disc formation. We first compared post-hatching development inside agarose tunnels or free-floating over an agarose-coated dish in serum- and glucose-enriched medium (PHD medium). Culture inside agarose tunnels shaped embryo morphology by physical constriction, but it restricted embryo growth and did not provide any significant advantage in terms of development of hypoblast and epiblast lineages. In contrast to PHD medium, a chemically defined and enriched medium (N2B27) supported complete hypoblast migration and epiblast survival in vitro, even in the absence of agarose coating. Cells expressing the pluripotency marker SOX2 were observed in ~56% of the embryos and ~25% developed embryonic disc-like structures formed by SOX2+ cells. In summary, here we provide a culture system that supports trophectoderm proliferation, hypoblast migration and epiblast survival after the blastocyst stage.

180 FATTY ACID COMPOSITION IN FOLLICULAR FLUID OF PREPUBERTAL GOAT OVARIES IN WINTER AND AUTUMN

2015 ◽  
Vol 27 (1) ◽  
pp. 181
Author(s):  
M. T. Paramio ◽  
M. Roura ◽  
S. Hammami ◽  
D. Izquierdo ◽  
M. G. Catalá
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Embryo Development ◽  
Follicular Fluid ◽  
Oocyte Quality ◽  
Blastocyst Stage ◽  
Omega 3 ◽  
Chromatography Analysis ◽  
Sperm Penetration

Fatty acids (FA) in follicular fluid (FF) play an important role on oocyte quality and embryo development (Fouladi-Nashta et al. 2007 Biol. Reprod. 77, 9–17). In our laboratory, we have shown in prepubertal goat differences in the percentage of blastocysts produced in vitro according to season. Thus, we have found in winter 15.8% and in autumn a decrease up to 4.7% of blastocysts that were produced from oocytes of 1 month old suckling Murciano-Granadina goat females and IVF with fresh semen. The aim of this study was to analyse composition of FF in order to find an explanation to seasonal changes in in vitro embryo production. Ovaries were recovered in winter and autumn from 1 month suckling goats (Murciano-Granadina) from a local slaughterhouse and the FF of all visible follicles was recovered using a sterile syringe. Each sample containing a pool of FF of different ovaries was frozen at –80°C until chromatography analysis. For the FA analysis, the Sukhija and Palmquist (1988 J. Agric. Food Chem. 36, 1202–1206) protocol with some adaptations was used. Briefly, 200 μL of FF sample was vortexed for 60 s with 250 μL of toluene and 1 mL of HCL (5%) and then warmed in a water bath for 1 h at 70°C. Subsequently 1.25 mL of K2CO3 (12%) and 500 μL of toluene was added, vortexed for 30 s and centrifuged for 5 min (3000 rpm). Finally the supernatant was recovered and dried with Na2SO4. The extracted samples were maintained in –20°C until gas chromatographic analysis (123–2362, Agilent Technologies Inc., Santa Clara, CA). The results in Table 1 express the mean of 3 replicates of follicular fluid pool as micromolar concentration of FA in FF. The FA profile in FF showed significant higher concentrations of α-linolenic (C18:3n3), eicosapentaenoic (EPA), docosahexaenoic (DHA), and omega-3 (n-3 polyunsaturated fatty acid; PUFA) in winter compared to autumn. This could be indicating that these PUFA have a positive effect on oocyte quality because of the higher embryo development of these oocytes during winter. Studies in our laboratory have shown that sperm penetration and normal zygotes were similar in both seasons even though the blastocyst yield was statistically higher in winter. We can speculate that fatty acids in the follicular environment are affecting the oocyte quality, increasing the possibility of reaching the blastocyst stage in prepubertal goat according to season. Further studies should be done to reach a more accurate conclusion. Table 1.Concentration (µM) of fatty acids in FF of prepubertal goat during winter and autumn (3 replicates)

Butyryl- and acetylcholine-esterases in human IVF-derived follicular fluid and luteinizing granulosa cells

2013 ◽  
Vol 121 (03) ◽  
Author(s):  
J Blohberger ◽  
D Einwang ◽  
D Berg ◽  
U Berg ◽  
S Hecht ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid ◽  
Human Ivf

PCOS follicular fluid derived exosomal miR-424-5p induces granulosa cells senescence by targeting CDCA4 expression

2021 ◽  
pp. 110030
Author(s):  
Dong Yuan ◽  
Jing Luo ◽  
Yixuan Sun ◽  
Lijuan Hao ◽  
Jing Zheng ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Follicular Fluid

scholarly journals A rapid and robust method for the cryopreservation of human granulosa cells

2021 ◽  
Author(s):  
Sarah Beschta ◽  
Katja Eubler ◽  
Nancy Bohne ◽  
Ignasi Forne ◽  
Dieter Berg ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Large Scale ◽  
Ovarian Function ◽  
Oocyte Retrieval ◽  
Cell Contact ◽  
Cellular Model ◽  
Preovulatory Follicle ◽  
Human Granulosa Cells ◽  
Ovarian Cells

AbstractHuman primary granulosa cells (GCs) derived from women undergoing oocyte retrieval can be cultured and used as a cellular model for the study of human ovarian function. In vitro, they change rapidly, initially resembling cells of the preovulatory follicle and then cells of the corpus luteum. They are derived from individual patients, whose different medical history, lifestyle and age lead to heterogeneity. Thus, cells can rarely be ideally matched for cellular experiments or, if available, only in small quantities. We reasoned that cryopreservation of human GCs may be helpful to improve this situation. Previous studies indicated the feasibility of such an approach, but low survival of human GCs was reported, and effects on human GC functionality were only partially evaluated. We tested a slow freezing protocol (employing FCS and DMSO) for human GCs upon isolation from follicular fluid. We compared cryopreserved and subsequently thawed cells with fresh, non-cryopreserved cells from the same patients. About 80% of human GCs survived freezing/thawing. No differences were found in cell morphology, survival rate in culture, or transcript levels of mitochondrial (COX4, OPA1, TOMM20), steroidogenic (CYP11A1, CYP19A1) or cell–cell contact genes (GJA1) between the two groups in cells cultured for 1–5 days. A proteomic analysis revealed no statistically significant change in the abundance of a total of 5962 proteins. The two groups produced comparable basal levels of progesterone and responded similarly to hCG with elevation of progesterone. Taken together, our results show this to be a rapid and readily available method for the cryopreservation of human GCs. We anticipate that it will allow future large-scale experiments and may thereby improve cellular studies with human ovarian cells.

scholarly journals Analysis of Morphokinetic Parameters of Feline Embryos Using a Time-Lapse System

Animals ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 748
Author(s):  
Joanna Kochan ◽  
Agnieszka Nowak ◽  
Barbara Kij ◽  
Sylwia Prochowska ◽  
Wojciech Niżański
Keyword(s):  
Embryo Development ◽  
Embryo Quality ◽  
Time Lapse ◽  
Blastocyst Stage ◽  
Cavity Formation ◽  
Non Invasive ◽  
Morphokinetic Parameters ◽  
Morphological Defects ◽  
Development In Vitro

The aim of this study was to analyze the morphokinetic parameters of feline embryos using a time lapse system. Oocytes matured in vitro were fertilized (IVF) and in vitro cultured in a time lapse-system (Primo Vision®, Gothenburg, Sweden). The first cell division of embryos occurred between 17 h post insemination (hpi) and 38 hpi, with the highest proportion of embryos (46%) cleaving between 21 and 24 hpi. The timing of the first cleavage significantly affected further embryo development, with the highest development occurring in embryos that cleaved at 21–22 hpi. Embryos that cleaved very early (17–18 hpi) developed poorly to the blastocyst stage (2%) and none of the embryos that cleaved later than 27 hpi were able to reach the blastocyst stage. Morphological defects were observed in 48% of the embryos. There were no statistically significant differences between the timing intervals of the first cleavage division and the frequency of morphological defects in embryos. Multiple (MUL) morphological defects were detected in more than half (56%) of the abnormal embryos. The most frequent single morphological defects were cytoplasmic fragmentation (FR) (8%) and blastomere asymmetry (AS) (6%). Direct cleavage (DC) from 1–3 or 3–5 blastomeres, reverse cleavage (RC) and vacuoles were rarely observed (2–3%). The timing of blastocyst cavity formation is a very good indicator of embryo quality. In our study, blastocyst cavity formation occurred between 127–167 hpi, with the highest frequency of hatching observed in blastocysts that cavitated between 142–150 hpi. Blastocysts in which cavitation began after 161 h did not hatch. In conclusion, the timing of the first and second cleavage divisions, the timing of blastocyst cavity formation and morphological anomalies can all be used as early and non-invasive indicators of cat embryo development in vitro.

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