scholarly journals Oocyte-Secreted Factors Synergize With FSH to Promote Aromatase Expression in Primary Human Cumulus Cells

2018 ◽  
Vol 104 (5) ◽  
pp. 1667-1676 ◽  
Author(s):  
Elie Hobeika ◽  
Marah Armouti ◽  
Hamsini Kala ◽  
Michele A Fierro ◽  
Nicola J Winston ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Cumulus Cells ◽  
Aromatase Expression ◽  
Vitro Fertilization ◽  
Protein Levels ◽  
Morphogenetic Protein ◽  
Smad3 Phosphorylation ◽  
Igf1 Receptor ◽  
Stimulation Of

Abstract Context The role of growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) on aromatase regulation is poorly understood in humans. Objective Determine GDF9 and BMP15 effects on FSH stimulation of estradiol production in primary human cumulus granulosa cells (GCs). We hypothesized that the combination of GDF9 and BMP15 potentiates FSH-induced aromatase expression. Design Primary human cumulus GCs in culture. Setting University infertility center. Patients or Other Participants GCs of 60 women undergoing in vitro fertilization were collected. Interventions Cells were treated with GDF9 and/or BMP15 (GB) in the presence or absence of FSH, dibutyryl cAMP, or SMAD inhibitors. Main Outcome Measures Promoter activity, mRNA, protein, and estradiol levels were quantified. Results FSH and GB treatment increased CYP19A1 promoter activity, mRNA, and protein levels as well as estradiol when compared with cells treated with FSH only. GB treatment potentiated cAMP stimulation of aromatase and IGF2 stimulation by FSH. GB effects were inhibited by SMAD3 inhibitors and IGF1 receptor inhibitors. GB, but not FSH, stimulates SMAD3 phosphorylation. Conclusion The combination of GDF9 and BMP15 potently stimulates the effect of FSH and cAMP on CYP19a1 promoter activity and mRNA/protein levels. These effects translate into an increase in estradiol production. This potentiation seems to occur through activation of the SMAD2/3 and SMAD3 signaling pathway and involves, at least in part, the effect of the IGF system.

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.

scholarly journals A Brief Incubation of Cumulus-Enclosed Mouse Eggs in a Calcium-Free Medium Containing a High Concentration of Calcium-Chelator Markedly Improves Preimplantation Development

2020 ◽  
Vol 17 (10) ◽  
pp. 3505
Author(s):  
Valeria Merico ◽  
Silvia Garagna ◽  
Maurizio Zuccotti
Keyword(s):  
In Vitro Fertilization ◽  
Mammalian Species ◽  
Developmental Rate ◽  
Cumulus Cells ◽  
Fertilization Rate ◽  
Preimplantation Development ◽  
High Concentration ◽  
Vitro Fertilization ◽  
Positive Effects

The presence of cumulus cells (CCs) surrounding ovulated eggs is beneficial to in vitro fertilization and preimplantation development outcomes in several mammalian species. In the mouse, this contribution has a negligible effect on the fertilization rate; however, it is not yet clear whether it has positive effects on preimplantation development. Here, we compared the rates of in vitro fertilization and preimplantation development of ovulated B6C3F1 CC-enclosed vs. CC-free eggs, the latter obtained either after a 5 min treatment in M2 medium containing hyaluronidase or after 5–25 min in M2 medium supplemented with 34.2 mM EDTA (M2-EDTA). We found that, although the maintenance of CCs around ovulated eggs does not increment their developmental rate to blastocyst, the quality of the latter is significantly enhanced. Most importantly, for the first time, we describe a further quantitative and qualitative improvement, on preimplantation development, when CC-enclosed eggs are isolated from the oviducts in M2-EDTA and left in this medium for a total of 5 min prior to sperm insemination. Altogether, our results establish an important advancement in mouse IVF procedures that would be now interesting to test on other mammalian species.

scholarly journals The Role of Interleukin-23 in the Early Development of Emphysema in HIV1+Smokers

2016 ◽  
Vol 2016 ◽  
pp. 1-14 ◽  
Author(s):  
Igor Z. Barjaktarevic ◽  
Ronald G. Crystal ◽  
Robert J. Kaner
Keyword(s):  
Tissue Destruction ◽  
Epithelial Lining Fluid ◽  
Protein Levels ◽  
Knowing That ◽  
Lung Epithelial ◽  
Interleukin 23 ◽  
Metalloproteinase 9 ◽  
Stimulation Of

Rationale.Matrix metalloproteinase-9 (MMP-9) expression is upregulated in alveolar macrophages (AM) of HIV1+smokers who develop emphysema. Knowing that lung epithelial lining fluid (ELF) of HIV1+smokers contains increased levels of inflammatory cytokines compared to HIV1−smokers, we hypothesized that upregulation of lung cytokines in HIV1+smokers may be functionally related to increased MMP-9 expression.Methods.Cytokine arrays evaluated cytokine protein levels in ELF obtained from 5 groups of individuals: HIV1−healthy nonsmokers, HIV1−healthy smokers, HIV1−smokers with low diffusing capacity (DLCO), HIV1+nonsmokers, and HIV1+smokers with lowDLCO.Results. Increased levels of the Th17 related cytokine IL-23 were found in HIV1−smokers with lowDLCOand HIV1+smokers and nonsmokers. Relative IL-23 gene expression was increased in AM of HIV1+individuals, with greater expression in AM of HIV1+smokers with lowDLCO. Infection with HIV1in vitroinduced IL-23 expression in normal AM. IL-23 stimulation of AM/lymphocyte coculturesin vitroinduced upregulation of MMP-9. Lung T lymphocytes express receptor IL-23R and interact with AM in order to upregulate MMP-9.Conclusion. This mechanism may contribute to the increased tissue destruction in the lungs of HIV1+smokers and suggests that Th17 related inflammation may play a role.

Ligand-dependent, Pit-1/growth hormone factor-1 (GHF-1)-independent transcriptional stimulation of rat growth hormone gene expression by thyroid hormone receptors in vitro

1993 ◽  
Vol 13 (3) ◽  
pp. 1719-1727
Author(s):  
C S Suen ◽  
W W Chin
Keyword(s):  
Gene Expression ◽  
Growth Hormone ◽  
Thyroid Hormone ◽  
Promoter Activity ◽  
Hormone Receptors ◽  
Nuclear Extract ◽  
In Vitro Transcription ◽  
Stimulation Of ◽  
Transcriptional Stimulation

The expression of the rat growth hormone (rGH) gene in the anterior pituitary gland is modulated by Pit-1/GHF-1, a pituitary-specific transcription factor, and by other more widely distributed factors, such as the thyroid hormone receptors (TRs), Sp1, and the glucocorticoid receptor. Thyroid hormone (T3)-mediated transcriptional stimulation of rGH gene expression has been extensively studied in vivo and in vitro including the measurements of (i) rGH mRNA by blot hybridization, (ii) transcriptional rate of rGH gene by nuclear run-on, and (iii) reporter gene expression in which a chimeric plasmid containing 5'-flanking sequences of the rGH gene linked to a reporter gene has been transfected either stably or transiently into pituitary and/or nonpituitary cells. From these studies, it has been suggested that the Pit-1/GHF-1 binding site is necessary for full T3 action. We developed a cell-free in vitro transcription system to examine further the roles of the TRs and Pit-1/GHF-1 in rGH gene activation. Using GH3 nuclear extract as a source of TRs and Pit-1/GHF-1, this in vitro transcription assay showed that T3 stimulation of rGH promoter activity is dependent on the addition of T3 to the GH3 nuclear extract. This transcriptional stimulation was augmented with increasing concentrations of ligand and was T3, but not T4 or reverse T3, specific. T3-mediated stimulation of rGH promoter activity was completely abolished by preincubation of the nuclear extract with rGH-thyroid hormone response element (-200 to -160) but not with Pit-1/GHF-1 (-137 to -65) oligonucleotides. Further, neither deletion of both Pit-1/GHF-1 binding sites nor mutation of the proximal Pit-1/GHF-1 binding site from the rGH promoter abrogated the T3 effect. These results provide evidence that T3-stimulated rGH promoter activity is independent of Pit-1/GHF-1 and raise the possibility that the stimulation of rGH gene expression by T3 might involve direct interaction of TRs with the general transcriptional apparatus.

scholarly journals Regulation of AMH by oocyte-specific growth factors in human primary cumulus cells

Reproduction ◽  
2017 ◽  
Vol 154 (6) ◽  
pp. 745-753 ◽  
Author(s):  
Scott Convissar ◽  
Marah Armouti ◽  
Michelle A Fierro ◽  
Nicola J Winston ◽  
Humberto Scoccia ◽  
...  
Keyword(s):  
Fold Increase ◽  
Cumulus Cells ◽  
Maximal Effect ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Protein Levels ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Signaling Inhibitors

The regulation of AMH production by follicular cells is poorly understood. The purpose of this study was to determine the role of the oocyte-secreted factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on AMH production in primary human cumulus cells. Cumulus cells from IVF patients were cultured with a combination of GDF9, BMP15, recombinant FSH and specific signaling inhibitors. Stimulation with GDF9 or BMP15 separately had no significant effect onAMHmRNA levels. In contrast, simultaneous stimulation with GDF9 and BMP15 (G + B) resulted in a significant increase inAMHmRNA expression. Increasing concentration of G + B (0.6, 2.5, 5 and 10 ng/mL) stimulated AMH in a dose-dependent manner, showing a maximal effect at 5 ng/mL. Western blot analyses revealed an average 16-fold increase in AMH protein levels in cells treated with G + B when compared to controls. FSH co-treatment decreased the stimulation of AMH expression by G + B. The stimulatory effect of G + B on the expression of AMH was significantly decreased by inhibitors of the SMAD2/3 signaling pathway. These findings show for the first time that AMH production is regulated by oocyte-secreted factors in primary human cumulus cells. Moreover, our novel findings establish that the combination of GDF9 + BMP15 potently stimulates AMH expression.

scholarly journals A Newly Authenticated Compound from Traditional Chinese Medicine Decoction Induces Melanogenesis in B16-F10 Cells by Increasing Tyrosinase Activity

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Xiu Juan Xin ◽  
Jiahong Zou ◽  
Tao Zou ◽  
Huoli Shang ◽  
Li Yun Sun
Keyword(s):  
Chinese Medicine ◽  
Traditional Chinese Medicine ◽  
Cell Number ◽  
Tyrosinase Activity ◽  
Chinese Traditional Medicine ◽  
Inhibition Rate ◽  
Disease Treatment ◽  
Protein Levels ◽  
Stimulation Of

Vitiligo is a kind of skin dysfunction on melanogenesis. The highly prevalent, chronic, and distinctive complexion changes on patients have imposed enormous psychic and economic burden on both individuals and society. Traditional Chinese Medicine (TCM) is a kind of precious source on chronic disease treatment, including skin dysfunctional diseases. In our previous study, a new compound named apigenin-7-butylene glucoside has been authenticated and purified from a prescription of Chinese traditional medicine formula which has been used clinically in vitiligo treatment. The aim of this work is to evaluate the effects of this compound on melanogenesis using melanoma cell B16-F10 in vitro. The results showed that apigenin-7-butylene glucoside had almost no cytotoxicity on B16-F10 cells within a lower dose of 5.0 μg ml-1 and enhanced the melanin level to about 41% and tyrosinase activity to 1.32-fold when compared with controls. The compound showed minor cytotoxicity to B16-F10 cells at the higher concentration of 10 μg ml-1 and 50 μg ml-1, the inhibition rate was 8.4% and 11.8%, and the melanin level and tyrosinase activity showed a decreased trend because of the lower cell number at the higher concentrations. The results indicated that apigenin-7-butylene glucoside was safe to B16-F10 cells within a lower concentration, <5.0 μg ml-1. Incubated with 5.0 ug ml-1of apigenin-7-butylene glucoside for 48 hours, the mRNA and protein levels of Tyr, Trp-1, and Trp-2 genes were all increased except Mitf in B16-F10 cells. The stimulation of apigenin-7-butylene glucoside on melanogenesis of B16-F10 cells through Tyr, Trp-1, and Trp-2 pathway highlighted the potential usage of the compound in vitiligo treatment.

scholarly journals Features of the preparation of biological material for genome editing in cattle

2019 ◽  
Vol 191 (12) ◽  
pp. 40-44
Author(s):  
A. Barkova ◽  
M. Modorov ◽  
G. Isaeva ◽  
A. Krivonogova
Keyword(s):  
Genome Editing ◽  
Biological Material ◽  
Genetic Material ◽  
Cumulus Cells ◽  
Vitro Fertilization ◽  
Expected Time ◽  
Donor Material ◽  
Material Transportation

Abstract. To carry out genome editing in cattle, an effective and well-functioning system for obtaining gametes, fertilizing eggs and their cryopreservation is necessary. Aim of the work: review and research of present-day existing methods of obtaining, insemination and cryopreservation of donor material, in order to provide genome editing in cows. Methods and materials. The work is completed according to the theme No. 0532-2019-0001 “Development of complex technology of marker-based genome selection of agricultural animals” within State Order of Ministry of Education and Science of the Russian Federation. The analysis of open scientific literature on the issues of in vitro fertilization in animals, cryopreservation of oocytes and embryons, sperm preparation and methods of insemination of cows’ oocytes, and cryopreservation of oocytes and embryons of animals is done. Features of the preparation of biological material of cattle for genome editing by microinjection into ooplasm are described. Results of research and duscussion. At present time there are two ways to obtain donor material from cattle: from live animals and taking ovaries after slaughtering cows. Material transportation is carried out at a temperature of 30–37 °C depending on the distance to the laboratory and expected time period of transportation. Oocyte-cumulus complexes can be removed by ovarian dissection and aspiration of visible follicles. In both cases, immature eggs are predominantly obtained. Subsequent ripening is carried out in vitro using special media in a CO2 incubator. The culture medium for oocyte maturation should contain hormones that mimic the peak of LH (luteinizing hormone), which occurs in vivo during the maturation of oocytes before ovulation. To accumulate a certain number of eggs at the stage of MII, it is recommended to carry out their cryopreservation by the method of vitrification, having previously released the oocyte from the cumulus cells. After thawing, oocytes need to be incubated for 2–3 hours 38.5 °C in 5–6.5% CO2 to restore the spindle. In order to make editing more effective, the introduction of genetic material is recommended to be carried out in parallel with the fertilization method “icsi”. In humans, mice and rabbits, an injection of sperm into the cytoplasm is sufficient to activate the oocyte, however, in cattle, just micro-injection of the sperm is not enough and often the male pronucleus does not form. To solve the problem, various methods are used, including freezing-thawing of sperm, resulting in damage of a membrane, or addition of heparin-glutathione into the medium that increases decondensation of the sperm DNA.

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