Evaluating the Effects of Different Concentrations of Human Follicular Fluid on Growth, Development, and PCNA Gene Expression of Mouse Ovarian Follicles

2020 ◽  
Vol 209 (2–3) ◽  
pp. 75-82
Author(s):  
Negar Molaeeghaleh ◽  
Shahriyar Tork ◽  
Shabnam Abdi ◽  
Shabnam Movassaghi
Keyword(s):  
Gene Expression ◽  
Survival Rate ◽  
Follicular Fluid ◽  
Ovarian Follicles ◽  
Nuclear Antigen ◽  
Nmri Mouse ◽  
Human Follicular Fluid ◽  
Follicle Culture ◽  
Growth Development

Follicle culture in vitro provides a method for investigating stages of folliculogenesis that can lead to preserving fertility through cryopreservation techniques. This study aims to assess the effects of various concentrations of human follicular fluid (hFF) on growth, development, and expression of the proliferating cell nuclear antigen (PCNA) gene in mouse ovarian follicles in vitro. Preantral follicles were isolated from 14-day NMRI mouse ovaries. The follicles were cultured in basic media enriched with FBS, FSH, and insulin-transferrin-selenium, and supplemented with different concentrations of hFF (10, 20, and 30%) for 12 days. During the culture period, survival rate and follicular maturation, follicular diameter, levels of estrogen and progesterone secretion, and PCNA gene expression rate were evaluated. Survival rate, maturation, and antrum formation were significantly higher in the 10% hFF group than in the 20 and 30% hFF groups. On day 4, follicle diameter in the 10% hFF group was also higher than in the 20 and the 30% hFF group. In comparison with other groups, significantly higher estrogen and progesterone production levels were measured in the 10% hFF group. PCNA gene expression was also higher with 10 than 20 and 30% hFF concentrations. The present study suggests that addition of 10% hFF to mice ovarian preantral follicle culture media enhances follicle growth and oocyte maturation.

scholarly journals Regulation of gene expression in endothelial cells: the role of human follicular fluid

2005 ◽  
Vol 34 (1) ◽  
pp. 37-46 ◽  
Author(s):  
R Gruemmer ◽  
L Klein-Hitpaß ◽  
J Neulen
Keyword(s):  
Gene Expression ◽  
Endothelial Cells ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Regulation Of Gene Expression ◽  
Angiogenic Factors ◽  
Human Follicular Fluid ◽  
Angiopoietin 2

A precise regulation of angiogenesis is a prerequisite for an adequate maturation of ovarian follicles. Despite the production of vascular endothelial growth factor (VEGF) by granulosa cells in antral follicles, angiogenesis is restricted to the theca cell layer. The maturing follicle remains avascular before ovulation, implying regulatory mechanisms which prevent premature follicular vascularization. In order to investigate the role of follicular fluid and of granulosa cells in the regulation of endothelial gene expression, human umbilical vein endothelial cells (HUVECs) were incubated in vitro with media conditioned with human follicular fluid obtained from individual patients undergoing oocyte retrieval for in vitro fertilization procedures or with culture medium conditioned by human granulosa cells respectively. Using microarray technology, the gene expression pattern was compared between untreated monolayers of HUVECs and HUVECs treated either with follicular fluid or with granulosa cell conditioned media. We identified a total of 15 genes that were significantly up-regulated and 11 genes that were significantly down-regulated in endothelial cells treated with follicular fluid at least 2.5-fold in more than 70% of comparisons. Up-regulated genes involved in angiogenesis were the anti-angiogenic factors gro-beta (16.5-fold), angiopoietin-2 (3.9-fold), alpha-2-macroglobulin (24.3-fold) and the pro-angiogenic factors E-selectin (5.3-fold) and vascular cell adhesion molecule-1 (VCAM-1) (4.4-fold), whereas a significant down-regulation of the pro-angiogenic genes fibulin-5 (3.5-fold) and elastin (14.9-fold) could be observed. Culturing of HUVECs with conditioned medium from cultured human luteinized granulosa cells demonstrated a similar regulatory pattern of gene expression for fibulin-5, elastin, gro-beta, and E-selectin. The gene regulation in endothelial cells by follicular fluid could be confirmed by RT-PCR for gro-beta, angiopoietin-2, elastin, fibulin-5, and E-selectin. The present work reveals that compounds secreted by granulosa cells lead to the expression of anti-angiogenic factors on the transcript level in endothelial cells and thus could help to explain the temporal and spatial discrepancy between the high expression of VEGF and the restricted angiogenesis in the preovulatory follicle.

scholarly journals Relaxin protein and gene expression in ovarian follicles of immature pigs

1998 ◽  
Vol 21 (2) ◽  
pp. 179-187 ◽  
Author(s):  
KM Ohleth ◽  
Q Zhang ◽  
CA Bagnell
Keyword(s):  
Gene Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Ovarian Follicle ◽  
Follicular Development ◽  
Ovarian Follicles ◽  
In Vitro Growth ◽  
Follicle Size ◽  
Theca Interna

Relaxin production by the ovarian follicle of gonadotropin-primed, prepubertal gilts is well documented. As far as we are aware, a source of relaxin in pig follicles, independent of gonadotropins, has not yet been reported. Therefore, the objective of this study was to determine whether relaxin is produced in porcine follicles in the absence of exogenous or cyclic gonadotropins. In immature pigs, immunoreactive relaxin was detected in fluids from small (1-3 mm), medium (4-5 mm) and large (>6 mm) follicles and localized to the theca interna of large follicles. Relaxin levels in follicular fluid significantly increased with follicle size (P<0.05). Relaxin mRNA was detected in whole small- and medium-sized follicles. In large follicles, the relaxin gene was expressed in thecal layers, but not granulosa cells. The abundance of relaxin transcript did not change with follicle size. In summary, relaxin protein and mRNA were detected in porcine follicles from immature animals, indicating that relaxin is produced in the porcine follicle in the absence of exogenous or cyclic gonadotropins. Relaxin's in vitro growth effects on porcine granulosa and theca cells support this follicular relaxin as a growth modulator during porcine follicular development.

scholarly journals Signalling pathways and mechanistic cues highlighted by transcriptomic analysis of primordial, primary, and secondary ovarian follicles in domestic cat

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shauna Kehoe ◽  
Katarina Jewgenow ◽  
Paul R. Johnston ◽  
Susan Mbedi ◽  
Beate C. Braun
Keyword(s):  
Gene Expression ◽  
Transforming Growth Factor ◽  
Ovarian Follicle ◽  
Mammalian Species ◽  
Expression Patterns ◽  
Ovarian Follicles ◽  
Domestic Cat ◽  
Transcriptional Analysis ◽  
Ovarian Folliculogenesis

AbstractIn vitro growth (IVG) of dormant primordial ovarian follicles aims to produce mature competent oocytes for assisted reproduction. Success is dependent on optimal in vitro conditions complemented with an understanding of oocyte and ovarian follicle development in vivo. Complete IVG has not been achieved in any other mammalian species besides mice. Furthermore, ovarian folliculogenesis remains sparsely understood overall. Here, gene expression patterns were characterised by RNA-sequencing in primordial (PrF), primary (PF), and secondary (SF) ovarian follicles from Felis catus (domestic cat) ovaries. Two major transitions were investigated: PrF-PF and PF-SF. Transcriptional analysis revealed a higher proportion in gene expression changes during the PrF-PF transition. Key influencing factors during this transition included the interaction between the extracellular matrix (ECM) and matrix metalloproteinase (MMPs) along with nuclear components such as, histone HIST1H1T (H1.6). Conserved signalling factors and expression patterns previously described during mammalian ovarian folliculogenesis were observed. Species-specific features during domestic cat ovarian folliculogenesis were also found. The signalling pathway terms “PI3K-Akt”, “transforming growth factor-β receptor”, “ErbB”, and “HIF-1” from the functional annotation analysis were studied. Some results highlighted mechanistic cues potentially involved in PrF development in the domestic cat. Overall, this study provides an insight into regulatory factors and pathways during preantral ovarian folliculogenesis in domestic cat.

Antioxidant activities and lipid peroxidation status in human follicular fluid: age-dependent change

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
H. Debbarh ◽  
N. Louanjli ◽  
S. Aboulmaouahib ◽  
M. Jamil ◽  
L. Ahbbas ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Follicular Fluid ◽  
Maternal Age ◽  
Antioxidant Activities ◽  
Human Follicular Fluid ◽  
Age Related ◽  
Group I ◽  
Group Ii ◽  
Scavenging Efficiency

Summary Maternal age is a significant factor influencing in vitro fertilization (IVF) outcomes. Oxidative stress (OS) is one of the major causes of age-related cellular and molecular damage. The purpose of this work was to investigate the correlation between maternal age with intrafollicular antioxidants and OS markers in follicular fluid (FF), and also to determine the OS status in patients of advanced age. This study was a prospective study including 201 women undergoing IVF whose age was between 24 and 45 years old. FF samples were obtained from mature follicles at the time of oocyte retrieval. After treatment of FF, lipid peroxidation levels (MDA) and enzyme activities such as superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glutathione (GSH) level were evaluated using spectrophotometry. The results indicated that the age cutoff point for increasing the MDA level was fixed at 37 years, allowing the study to be differentiated into two age groups. Group I included patients whose age was less than 37 years, and group II included patients whose age was greater than or equal 37 years. Statistical analysis revealed that MDA and GSH levels and GR activity were significantly higher in group II compared with group I. The SOD and CAT activities were significantly less in group II compared with group I. We concluded that from 37 years old a reproductive ageing was accompanied by a change in the antioxidant pattern in FF that impaired reactive oxygen species scavenging efficiency.

scholarly journals Expression of mRNAs for Pro-and Anti-Apoptotic Factors in Granulosa Cells and Follicular Fluid of Women Undergoing in Vitro Fertilization. A Pilot Study

2021 ◽  
Author(s):  
Jozsef Bodis ◽  
Endre Sulyok ◽  
Akos Varnagy ◽  
Viktória Prémusz ◽  
Krisztina Godony ◽  
...  
Keyword(s):  
Gene Expression ◽  
In Vitro Fertilization ◽  
Mrna Expression ◽  
Granulosa Cells ◽  
Follicular Fluid ◽  
Vitro Fertilization ◽  
Expression Levels ◽  
The Impact ◽  
Apoptotic Factors

Abstract BackgroundThis observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).Methods GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3±5.8 years, body mass index: 24.02±3.12 kg/m2, duration of infertility: 4.2±2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480. Results No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r=0.393, p=0.029), but the day of embryo transfer was negatively associated with GC LHR (r=-0.414, p=0.020) and GC FSHR transcripts (r=-0.535, p=0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.ConclusionOur study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

Extracellular vesicles of follicular fluid from heat-stressed cows modify the gene expression of in vitro-matured oocytes

2019 ◽  
Vol 205 ◽  
pp. 94-104 ◽  
Author(s):  
Felipe Morales Dalanezi ◽  
Henry David Mogollon Garcia ◽  
Rodrigo de Andrade Ferrazza ◽  
Fernanda Fagali Franchi ◽  
Patricia Kubo Fontes ◽  
...  
Keyword(s):  
Gene Expression ◽  
Extracellular Vesicles ◽  
Follicular Fluid

scholarly journals 24 Validation of swine enteroids as a model to study Lawsonia intracellularis pathogenesis

2019 ◽  
Vol 97 (Supplement_3) ◽  
pp. 21-22
Author(s):  
Ramya Lekha Medida ◽  
Talita P Resende ◽  
Connie Gebhart ◽  
Milena Saqui-Salces
Keyword(s):  
Gene Expression ◽  
Intestinal Epithelium ◽  
Culture Media ◽  
Paneth Cell ◽  
Nuclear Antigen ◽  
Atmospheric Conditions ◽  
Lawsonia Intracellularis ◽  
Swine Industry ◽  
Fatty Acid Binding

Abstract Lawsonia intracellularis is an obligate intracellular bacterium that causes proliferative ileitis, an enteric disease that costs more than $100 million annually to the US swine industry. Information about L. intracellualris pathogenesis is scarce due to lack of suitable in vitro models to study the infection-induced proliferative changes. L. intracellularis infection of the swine ileum has been shown to decrease the number of mucin-producing goblet cells and increase cell proliferation along with amplification of transient amplifying cells demonstrated by the expression of SOX9. The objective of this study was to validate the use of swine enteroids (three dimensional structures derived from adult intestinal stem cells) as a model to study the intestinal epithelial changes caused by L. intracellularis infection. Swine enteroids plated on transwell plate inserts to cover the surface area were infected with 108 L. intracellularis organisms in culture media per well and incubated at 37⁰C with atmospheric conditions of 8.0% oxygen, 8.8% carbon dioxide, and 83.2% nitrogen. Infected enteroids were collected after 7 days and gene expression levels of mucin 2 (MUC2), enterocyte marker fatty acid binding protein (FABP), endocrine marker chromogranin A (CGA), intestinal epithelium marker villin 1 (VIL1), paneth cell marker lysozyme (LYZ), proliferating cell nuclear antigen (PCNA) and SOX9 were measured. The expression of FABP and LYZ in L. intracellularis infected swine enteroids decreased by 50% and 20% respectively and the expression of SOX9 increased by 50%, with no significant changes for the levels of VIL1 and CGA. The changes observed in the infected swine enteroids reflect the profile of gene expression observed in L. intracellularis infected ileal tissue. Therefore, swine enteroids are a suitable in vitro system to study the dynamics of cell differentiation and proliferation of the intestinal epithelium induced by L. intracellularis infection.

scholarly journals In vitro ovarian follicle growth: a comprehensive analysis of key protocol variables†

2020 ◽  
Vol 103 (3) ◽  
pp. 455-470
Author(s):  
Leah E Simon ◽  
T Rajendra Kumar ◽  
Francesca E Duncan
Keyword(s):  
Wildlife Conservation ◽  
Culture Media ◽  
Ovarian Follicle ◽  
Three Dimensional ◽  
Ovarian Follicles ◽  
Culture Methods ◽  
Growth And Survival ◽  
Follicle Culture ◽  
Culture Techniques

Abstract Folliculogenesis is a complex process that requires integration of autocrine, paracrine, and endocrine factors together with tightly regulated interactions between granulosa cells and oocytes for the growth and survival of healthy follicles. Culture of ovarian follicles is a powerful approach for investigating folliculogenesis and oogenesis in a tightly controlled environment. This method has not only enabled unprecedented insight into the fundamental biology of follicle development but also has far-reaching translational applications, including in fertility preservation for women whose ovarian follicles may be damaged by disease or its treatment or in wildlife conservation. Two- and three-dimensional follicle culture systems have been developed and are rapidly evolving. It is clear from a review of the literature on isolated follicle culture methods published over the past two decades (1980–2018) that protocols vary with respect to species examined, follicle isolation methods, culture techniques, culture media and nutrient and hormone supplementation, and experimental endpoints. Here we review the heterogeneity among these major variables of follicle culture protocols.

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