P–596 Association of oleic acid production in cumulus-granulosa cells with glutathione of in vitro matured oocytes

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
S Fayezi ◽  
M Ghaffar. Novin ◽  
M Norouzian ◽  
M Nouri ◽  
L Farzadi
Keyword(s):  
Oleic Acid ◽  
Fatty Acid ◽  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Acid Production ◽  
In Vitro Maturation ◽  
Acid Synthesis ◽  
Glutathione Level ◽  
Metaphase Ii Oocytes

Abstract Study question Does oleic acid production in cumulus-granulosa cells affect glutathione levels of in vitro matured oocytes? Summary answer Oleic acid availability in cumulus-granulosa cells is associated with a higher glutathione level in in vitro matured oocytes. What is known already The monounsaturated fatty acid oleic acid is de novo synthesized by desaturation of stearic acid and can promote steroidogenesis and oocyte development in vitro. The endogenous antioxidant glutathione content in metaphase II oocyte is significantly higher than immature stages and is related to the normal oocyte maturation. Study design, size, duration Mouse germinal vesicles were co-cultured for 24 hours, during in vitro maturation, with granulosa cells treated with a specific inhibitor of oleic acid synthesis alone or in combination with oleic acid. Participants/materials, setting, methods Fluorescence staining was used to assess the glutathione content of mouse metaphase II oocytes following in vitro maturation as an indicator of cytoplasmic maturation. Glutathione was stained using Cell Tracker Blue –CMAC for 30 min at 37 °C. After being washed in fresh media, stained oocytes were photographed by a fluorescence microscope. Cell area and associated fluorescence were quantified in 20 metaphase II mouse oocytes randomly chosen from in vitro matured oocytes for each condition. Main results and the role of chance The intracellular glutathione content was profoundly lower in metaphase II oocytes obtained from co-cultures with inhibitor-treated cumulus-granulosa cells than with the control cumulus cells (–50%, p < 0.01). Oleic acid effectively recovered the negative effect of inhibitor on glutathione level nearly up to the level of the mock-treated cells. Limitations, reasons for caution The findings are limited to metaphase II. Measurement at more advanced stages of oocyte development is of interest. Inhibition of cellular fatty acid synthesis was performed solely with a specific chemical. Wider implications of the findings: Involvement of the oleic acid availability for cumulus-granulosa cells in normal oocyte maturation may be of relevance in reproductive disorders, particularly in the pathological mechanism of impaired oogenesis. Trial registration number 400/3226

265 HYALURONAN SYNTHESIS ABILITY OF PORCINE CUMULUS - OOCYTE COMPLEXES DERIVED FROM SMALL FOLLICLES

2013 ◽  
Vol 25 (1) ◽  
pp. 280
Author(s):  
M. Nakakoji ◽  
H. Funahashi
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Cumulus Cell ◽  
Cumulus Cells ◽  
Cumulus Expansion ◽  
Elisa Kit ◽  
Porcine Oocyte ◽  
Post Hoc ◽  
Metaphase Ii Oocytes

The degree of cumulus expansion, an important step in oocyte maturation, of porcine cumulus–oocyte complexes (COC) derived from small follicles (SF: 1 to 2 mm in diameter) is known to be lower than those derived from middle follicles (MF: 3 to 6 mm in diameter). The objective of this study was to compare the abilities of hyaluronan (HA) synthesis of COC from SF and MF. Furthermore, the effect of oestradiol during pre-incubation of COC on proliferation of the cumulus cells was examined. Cumulus–oocyte complexes from SF and MF of porcine ovaries were cultured for in vitro maturation [IVM, in modified porcine oocyte medium (Yoshioka et al. 2008 J. Reprod. Dev. 54, 208–213) supplemented with 50 µM β-mercaptoethanol, 10 IU mL–1 of eCG, 10 IU mL–1 of hCG, and 1 mM dbcAMP for 20 h and then in the fresh medium without those supplements for another 24 h]. Hyaluronan production was quantified at 20 h after the start of IVM with a commercial HA-ELISA kit (20 COC/tube × 4 times). The number of cumulus cells was assessed 0 and 20 h after the start of IVM (50 COC × 4 times). Furthermore, proliferation of cumulus cells was examined after pre-culture of COC (n = 40 COC × 5 times) in modified porcine oocyte medium with various concentrations of oestradiol (0, 0.1, 1, and 10 ng mL–1) for 6 h. Statistical analyses of results from 4 to 5 replicated trials were performed by ANOVA with a Bonferroni-Dunn post-hoc test (significance, P < 0.05). The degree of cumulus expansion of COC from MF (n = 152) was higher than that of COC from SF (n = 156). The incidence of metaphase-II oocytes was significantly lower in COC from SF (n = 133; 48.9%) than in COC from MF (n = 148; 74.7%). The HA content of COC was higher in those from MF (20.8 µg/COC) than in those from SF (10.8 µg/COC), whereas the content per cumulus cell was not different because the numbers of cumulus cells at 0 and 20 h were also higher in COC (n = 200 in each group) from MF (3.0 × 103 and 3.3 × 103 cells, respectively) than from SF (2.0 × 103 and 2.5 × 103 cells, respectively). Cumulus cells proliferated significantly in the presence of oestradiol, regardless of the concentration, during pre-incubation for 6 h (2.5 to 2.8 × 103 cells), as compared with the oestradiol-free controls (2.2 × 103 cells). These results demonstrate that the different abilities of cumulus expansion between COC (n = 200 in each group) from SF and MF may be due to the number of cumulus cells per COC. Pre-incubation in the presence of oestradiol stimulates the proliferation of cumulus cells and may improve the oocyte maturation of COC derived from SF.

273 CONSEQUENCE OF HIGH NONESTERIFIED FATTY ACID CONCENTRATIONS DURING BOVINE OOCYTE IN VITRO MATURATION ON mRNA TRANSCRIPT ABUNDANCE OF BLASTOCYSTS

2011 ◽  
Vol 23 (1) ◽  
pp. 234
Author(s):  
V. Van Hoeck ◽  
P. Bermejo-Álvarez ◽  
D. Rizos ◽  
A. Gutierrez-Adan ◽  
S. Andries ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Oocyte Maturation ◽  
Transcript Abundance ◽  
Acid Synthesis ◽  
Negative Energy ◽  
Nonesterified Fatty Acid ◽  
Adaptation To Stress ◽  
Developmental Capacity ◽  
In Vitro Oocyte Maturation

In recent years, high nonesterified fatty acid (NEFA) concentrations have been intensively discussed as an important metabolic cue linking negative energy balance in high yielding dairy cows early postpartum to disappointing fertility. Previous research has shown that high NEFA concentrations during in vitro oocyte maturation not only induce significant negative effects on the developmental capacity of oocytes but also reduce the quality and viability of the subsequent embryos that do survive until 7 days post-insemination. Because NEFA are known to regulate gene expression, the objective of this study was to investigate the effect of NEFA exposure during bovine in vitro oocyte maturation on the relative transcript abundance in the resultant day 7 blastocysts. This study focused on key genes related to embryonic quality, apoptosis, adaptation to stress, metabolism, DNA methylation, growth factors, and fatty acid synthesis. During a serum-free maturation period of 24 h, bovine cumulus–oocyte complexes were exposed to maturation medium (0.75% BSA) supplemented with 1) physiological NEFA concentrations (control; 150 μM of total NEFA, i.e. oleic, stearic, and palmitic acid), 2) elevated stearic acid concentrations (HSA; 75 μM of stearic acid), and 3) elevated NEFA concentrations (HCOMBI; 425 μM of total NEFA). Following IVF using semen from a bull of proven fertility, zygotes were cultured in SOF (+5% of FCS) medium for 7 days. Studied mRNA transcripts were quantified by real-time quantitative RT-PCR. Experiments were conducted to determine levels of each transcript relative to H2AZ in every sample. Relative mRNA abundance differences among groups were analysed by one-way ANOVA. Significant increases (P < 0.05) in relative mRNA abundance of the HCOMBI embryos were found for genes related to DNA methylation (DNMT3A), growth factors (IGF2R), glucose transport (SLC2A1), and fatty acid synthesis (ACSL1 and ACACA) compared with control embryos. Furthermore, HCOMBI embryos revealed a significantly higher expression (P < 0.05) of IGF2R and ACSL1 than did HSA embryos. The level of transcripts of genes related to mitochondrial biogenesis (TFAM) and the adaptation to stress (MNSOD) also tended to be increased in HCOMBI embryos compared with control embryos (P = 0.09 and P = 0.10, respectively). In conclusion, there is ample evidence that embryos originating from oocytes matured under negative energy balance conditions show aberrant transcriptional activities. These results might improve our current understanding of the possible mechanisms through which NEFA exposure during oocyte maturation affects the developmental capacity, quality, and viability of the resultant embryo.

230. Altered matrix composition of cumulus oocyte complexes following in vitro maturation

2005 ◽  
Vol 17 (9) ◽  
pp. 90
Author(s):  
K. R. Dunning ◽  
C. X. Yeo ◽  
D. L. Russell
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
In Vitro Maturation ◽  
Full Length ◽  
Matrix Composition ◽  
In Vitro Fertilisation ◽  
Lh Surge ◽  
Matrix Expansion

The luteinizing hormone (LH) surge initiates cumulus expansion, through synthesis of hyaluronan and cross-linking proteins including versican, which stabilise the cumulus oocyte complex (COC) matrix. Versican is a substrate for the protease ADAMTS-1 and mRNA for each are localised to granulosa cells (GCs) and greatly induced following the LH surge. In humans, the use of in vitro maturation (IVM) of oocytes is an appealing option, reducing costs and risk of side effects associated with in vitro fertilisation. IVM oocytes are of poorer quality, likely resulting from altered gene expression and environmental conditions during oocyte maturation. Real-time PCR showed that IVM and immature COCs from Balb/c mice have 12 and 13 fold-reduced levels of ADAMTS-1 and versican expression respectively compared to in vivo matured COCs (PMSG+hCG 12h). Ovulated COCs (PMSG+hCG 15h) had similar low levels of ADAMTS-1 and versican. Samples isolated from F1 C57Bl/6xCBA mice showed similar reduced versican and ADAMTS-1 mRNA. Western blot analysis revealed that full length and cleaved versican, from ADAMTS-1/4 activity, was not detected in immature COCs, was present in in vivo matured COCs isolated from follicles, but strongest in ovulated COCs. IVM COCs had no detectable versican protein, supporting the mRNA data. Full-length versican was also present in GCs after PMSG+hCG 12h or 15h. ADAMTS-1 protein was most abundant in in vivo matured COCs with reduced levels seen in ovulated COCs, but was absent from IVM and immature COCs. These results indicate that ADAMTS-1 and versican are secreted products of granulosa cells that bind and incorporate into the COC matrix. The presence of versican and ADAMTS-1 is not essential for cumulus matrix expansion in vitro, but may contribute to oocyte maturation, ovulation of the COC and/or interaction with sperm during fertilisation.

In vivo and in vitro effects of interleukin-1β on equine oocyte maturation and on steroidogenesis and prostaglandin synthesis in granulosa and cumulus cells

2009 ◽  
Vol 21 (2) ◽  
pp. 265 ◽  
Author(s):  
Maud Caillaud ◽  
Nadine Gérard
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
In Vitro Maturation ◽  
Cumulus Cells ◽  
Interleukin 1Β ◽  
Cox 2 ◽  
Group 2 ◽  
Cox 1

We analysed the effect of interleukin-1 on oocyte maturation and on steroid and prostaglandin production by equine granulosa and cumulus cells. In Experiment 1, interleukin-1β (IL-1β) was injected into the growing dominant follicle, which was punctured 38 h later. Follicular fluid was assayed for steroids and prostaglandin-F2α (PGF2α). Granulosa cells were analysed for 3β-hydroxysteroid dehydrogenase (3β-HSD), progesterone receptor (PR), cyclooxygenase 1 and 2 (Cox 1 and Cox 2) and steroidogenic acute regulatory protein (StAR) mRNAs. In Experiment 2, cumulus–oocyte complexes (COCs) were collected from slaughterhouse ovaries and cultured in different media: control group (TCM199 + BSA); Group 2 (+ IL-1β); Group 3 (+ EGF); Group 4 (+ EGF + IL-1β); and Group 5 (+ EGF + IL-1β + IL-1RA). Cumulus cells were analysed for 3β-HSD, PR, Cox 1, Cox 2 and StAR mRNAs. After injections of crude equine gonadotropin (CEG; LH effect) or IL-1β, progesterone and PGF2α levels increased, whereas 17β-oestradiol decreased. EGF induced an increase in the rate of in vitro maturation (P < 0.05), whereas IL-1β had a limited effect. IL-1β significantly decreased the rate of EGF-induced oocyte maturation (P < 0.05). Cox 2 mRNA level increases in granulosa cells after CEG injection (P = 0.07). In cumulus cells, StAR and PR mRNAs were lower in Group 2 and 3β-HSD mRNA was higher in Groups 4 and 5. These data confirm that IL-1 is involved in equine oocyte in vitro maturation. We demonstrated in vivo that IL-1β has an effect on steroids and PGF2α secretion in the preovulatory follicle.

scholarly journals The Differential Metabolomes in Cumulus and Mural Granulosa Cells from Human Preovulatory Follicles

2021 ◽  
Author(s):  
Er-Meng Gao ◽  
Bongkoch Turathum ◽  
Ling Wang ◽  
Di Zhang ◽  
Yu-Bing Liu ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Granulosa Cells ◽  
Quantitative Polymerase Chain Reaction ◽  
Cumulus Cells ◽  
Cholesterol Transport ◽  
Vitro Fertilization ◽  
Expression Of Genes ◽  
Preovulatory Follicles ◽  
Morphological Differences

AbstractThis study evaluated the differences in metabolites between cumulus cells (CCs) and mural granulosa cells (MGCs) from human preovulatory follicles to understand the mechanism of oocyte maturation involving CCs and MGCs. CCs and MGCs were collected from women who were undergoing in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) treatment. The differences in morphology were determined by immunofluorescence. The metabolomics of CCs and MGCs was measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) followed by quantitative polymerase chain reaction (qPCR) and western blot analysis to further confirm the genes and proteins involved in oocyte maturation. CCs and MGCs were cultured for 48 h in vitro, and the medium was collected for detection of hormone levels. There were minor morphological differences between CCs and MGCs. LC-MS/MS analysis showed that there were differences in 101 metabolites between CCs and MGCs: 7 metabolites were upregulated in CCs, and 94 metabolites were upregulated in MGCs. The metabolites related to cholesterol transport and estradiol production were enriched in CCs, while metabolites related to antiapoptosis were enriched in MGCs. The expression of genes and proteins involved in cholesterol transport (ABCA1, LDLR, and SCARB1) and estradiol production (SULT2B1 and CYP19A1) was significantly higher in CCs, and the expression of genes and proteins involved in antiapoptosis (CRLS1, LPCAT3, and PLA2G4A) was significantly higher in MGCs. The level of estrogen in CCs was significantly higher than that in MGCs, while the progesterone level showed no significant differences. There are differences between the metabolomes of CCs and MGCs. These differences may be involved in the regulation of oocyte maturation.

Collection and in vitro maturation of Mazama gouazoubira (brown brocket deer) oocytes obtained after ovarian stimulation

Zygote ◽  
2021 ◽  
pp. 1-7
Author(s):  
Luciana Diniz Rola ◽  
Eveline dos Santos Zanetti ◽  
Maite del Collado ◽  
Ellen de Fátima Carvalho Peroni ◽  
José Maurício Barbanti Duarte
Keyword(s):  
Oocyte Maturation ◽  
Ovarian Stimulation ◽  
In Vitro Maturation ◽  
Wildlife Conservation ◽  
Estradiol Benzoate ◽  
Ex Situ ◽  
Follicular Aspiration ◽  
Mazama Gouazoubira

Summary In vitro production of embryos has gained prominence as a tool for use in wildlife conservation programmes in situ and ex situ. However, the development of this technique depends on steps that include ovarian stimulation, collection and oocyte maturation. The purpose of this study was to assess the feasibility of an ovarian stimulation protocol for follicular aspiration, the efficiency of videolaparoscopy for follicular aspiration and test a medium for in vitro oocyte maturation for the species Mazama gouazoubira. Five females were submitted to repeated ovarian stimulation (hormone protocol using controlled internal drug release), and estradiol benzoate on D0 and eight injections of follicle-stimulating hormone, once every 12 h, from D4 onwards at 30-day intervals. Fourteen surgical procedures were performed in superstimulated females, resulting in the collection of 94 oocytes and an average of 17.1 ± 9.1 follicles observed, 13.5 ± 6.6 follicles aspirated and 7.2 ± 3.7 oocytes collected per surgery. After collection, the oocytes were submitted to in vitro maturation for 24 h and stained with Hoechst 33342 dye to evaluate their nuclear status; 64.5% of the oocytes reached MII and 16.1% were spontaneously activated by parthenogenesis. The nuclear status of oocytes that did not undergo in vitro maturation was evaluated; 80.9% were found to be immature.

scholarly journals Feasibility of Secondary Follicle Isolation, Culture and Achievement of In-Vitro Oocyte Maturation from Superovulated Ovaries: An Experimental Proof-of-Concept Study Using Mice

2021 ◽  
Vol 10 (13) ◽  
pp. 2757
Author(s):  
Xia Hao ◽  
Amandine Anastácio ◽  
Kenny A. Rodriguez-Wallberg
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Follicular Development ◽  
Clinical Situation ◽  
Experimental Proof ◽  
Secondary Follicle ◽  
17Β Estradiol ◽  
Follicle Size ◽  
And Control

Fertility preservation through ovarian stimulation, aiming at cryopreserving mature oocytes or embryos, is sometimes unsuccessful. This clinical situation deserves novel approaches to overcome infertility following cancer treatment in patients facing highly gonadotoxic treatment. In this controlled experimental study, we investigated the feasibility of in-vitro culturing secondary follicles isolated from superovulated ovaries of mice recently treated with gonadotropins. The follicle yields of superovulated ovaries were 45.9% less than in unstimulated controls. Follicles from superovulated ovaries showed faster growth pace during the initial 7 days of culture and secreted more 17β-estradiol by the end of culture vs controls. Parameters reflecting the outcome of follicular development and oocyte maturation competence in vitro were similar between superovulated and control groups, with a similar follicle size at the end of culture and around 70% survival. Nearly half of cultured follicles met the criteria for in-vitro maturation in both groups and approximately 60% of those achieved a mature MII oocyte, similarly in both groups. Over 60% of obtained MII oocytes displayed normal-looking spindle and chromosome configurations, without significant differences between the groups. Using a validated follicle culture system, we demonstrated the feasibility of secondary follicle isolation, in-vitro culture and oocyte maturation with normal spindle and chromosome configurations obtained from superovulated mice ovaries.

scholarly journals NUTRITIONAL FACTORS IN FATTY ACID SYNTHESIS BY TISSUE SLICES IN VITRO

1952 ◽  
Vol 197 (1) ◽  
pp. 181-191 ◽  
Author(s):  
Grace. Medes ◽  
Alice. Thomas ◽  
Sidney. Weinhouse
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acid Synthesis ◽  
Tissue Slices ◽  
Nutritional Factors
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