Steady-state level of epidermal growth factor (EGF) mRNA and effect of EGF on in vitro culture of caprine preantral follicles

2011 ◽  
Vol 344 (3) ◽  
pp. 539-550 ◽  
Author(s):  
Juliana Jales H. Celestino ◽  
Jamily B. Bruno ◽  
Márcia Viviane A. Saraiva ◽  
Rebeca M. P. Rocha ◽  
Ivina R. Brito ◽  
...  
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
In Vitro Culture ◽  
State Level ◽  
Steady State Level ◽  
Preantral Follicles ◽  
Epidermal Growth

scholarly journals Steady-state level of insulin-like growth factor-I (IGF-I) receptor mRNA and the effect of IGF-I on the in vitro culture of caprine preantral follicles

2012 ◽  
Vol 77 (1) ◽  
pp. 206-213 ◽  
Author(s):  
D.M. Magalhães-Padilha ◽  
A.B.G. Duarte ◽  
V.R. Araújo ◽  
M.V.A. Saraiva ◽  
A.P. Almeida ◽  
...  
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
In Vitro Culture ◽  
State Level ◽  
Steady State Level ◽  
Preantral Follicles ◽  
Factor I ◽  
Receptor Mrna ◽  
Igf I

118 EFFECTS OF EPIDERMAL GROWTH FACTOR ON IN VITRO SURVIVAL AND ANTRUM FORMATION OF ISOLATED OVINE PREANTRAL FOLLICLES

2014 ◽  
Vol 26 (1) ◽  
pp. 173 ◽  
Author(s):  
L. da Paz Santos ◽  
V. R. P. Barros ◽  
A. Y. P. Cavalcante ◽  
V. R. Araújo ◽  
M. H. T. Matos
Keyword(s):  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Basement Membrane ◽  
In Vitro Culture ◽  
Culture Medium ◽  
Formation Rate ◽  
Control Medium ◽  
Preantral Follicles ◽  
Epidermal Growth

The aim of this study was to verify the beneficial effects of different concentrations of epidermal growth factor (EGF) on survival and antrum formation of isolated ovine preantral follicles cultured in vitro. Ovine ovaries (n = 50) were collected from a local slaughterhouse and secondary follicles (150–200 μm in diameter), without antral cavities, were mechanically isolated by microdissection using 26-gauge needles. After selection, the follicles were individually cultured in 100-μL droplets of culture medium at 39°C and 5% CO2 in air for 18 days. The basic control medium consisted of α-minimal essential medium (α-MEM) supplemented with BSA; insulin, transferring and selenium; glutamine; hypoxanthine; and ascorbic acid and then referred to as α-MEM+. For the experimental conditions, follicles were cultured in α-MEM+ alone (control) or in different concentrations of EGF (1, 10, or 50 ng mL–1). Every other day, 60 μL of the culture media was replaced with fresh media. The morphological aspects of all ovine follicles were assessed every 6 days using a precalibrated ocular micrometer in a stereomicroscope at 100× magnification. Only those follicles showing an intact basement membrane, with bright and homogeneous granulosa cells and an absence of morphological signs of degeneration, were classified as morphologically normal follicles. The rupture of the basement membrane was also observed and characterised as follicle extrusion. In addition, antral cavity formation was defined as the emergence of a visible translucent cavity within the granulosa cell layers. Data from morphologically normal follicles, extruded follicles, and antrum formation rate during in vitro culture were expressed as percentages and compared by the chi-squared test, and differences were considered significant when P < 0.05. The results showed that the percentage of morphologically normal follicles decreased significantly throughout the culture periods in all the treatments, except in the 50 ng mL–1 EGF group, which maintained the percentage of normal follicles from Day 0 to 6. Considering the same culture period, 50 ng mL–1 EGF treatment significantly increased the percentage of morphologically normal follicles at Day 18 compared with the control group. Moreover, the addition of EGF to the culture medium, at 50 ng mL–1, significantly reduced the precocious extrusion of oocytes and increased the percentage of antrum formation compared with the control and 1 ng mL–1 EGF after 18 days of culture. Notably, there were no significant differences between 10 ng mL–1 EGF, control medium, and 1 ng mL–1 EGF treatments. In conclusion, this study demonstrated that the addition of EGF to the in vitro culture medium, at 50 ng mL–1, increased the proportion of morphologically normal follicles and antrum formation rate of isolated ovine preantral follicles. This work was supported by FACEPE (Process APQ-0705–5.05/10). L. P. Santos is a recipient of a grant from FACEPE (Brazil).

scholarly journals Steady-state level of messenger RNA and immunolocalization of aquaporins 3, 7, and 9 during in vitro growth of ovine preantral follicles

2015 ◽  
Vol 84 (1) ◽  
pp. 1-10 ◽  
Author(s):  
A.D. Sales ◽  
A.B.G. Duarte ◽  
G.Q. Rodrigues ◽  
L.F. Lima ◽  
G.M. Silva ◽  
...  
Keyword(s):  
Steady State ◽  
Messenger Rna ◽  
State Level ◽  
In Vitro Growth ◽  
Steady State Level ◽  
Preantral Follicles

311 ULTRASTRUCTURAL ANALYSIS OF OVINE PREANTRAL FOLLICLES CULTURED IN THE PRESENCE OF INDOLE ACETIC ACID, EGF, AND FSH

2006 ◽  
Vol 18 (2) ◽  
pp. 263
Author(s):  
E. Andrade ◽  
F. Landim-Alvarenga ◽  
J. Silva ◽  
M. Max ◽  
A. Alfieri ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Acetic Acid ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Indole Acetic Acid ◽  
Primordial Follicles ◽  
Preantral Follicles ◽  
Epidermal Growth ◽  
Cortical Slices

Previous studies from our team demonstrated that ovine primordial follicles are successfully activated in vitro after culturing in medium supplemented with 40 ng/mL indole acetic acid (IAA); besides that, the addition of IAA and epidermal growth factor (EGF) or EGF and follicle stimulating hormone (FSH) to the culture media were the most effective treatments to sustain the health and viability of activated ovine primordial follicles during in vitro culture. In this work, follicular quality was assessed only by histological studies; there is a great need to evaluate ultrastructural changes occurring in primordial follicles during activation in vitro. The aim of this study was to investigate the ultrastructural characteristics of preantral follicles after culturing of cortical slices in media containing IAA, EGF, and FSH, alone and in combination. Ovaries (n = 8) from adult merino ewes were collected at local slaughterhouses. Small pieces of ovarian fragments were removed for transmission electron microscopy (TEM). These pieces of ovarian cortex were cultured in culture dishes containing 1 mL aliquots of culture medium at 39�C with 5% CO2 in air. The media used were: (T1) Minimum essential medium (MEM) supplemented with ITS (insulin 6.25 �g/mL, transferrin 6.25 �g/mL and selenium 6.25 ng/mL), 0.23 mM pyruvate, 2 mM glutamine, 2 mM hypoxantine, 1.25 mg/mL bovine serum albumin (BSA), and antibiotics (100 ��g/mL penicillin and 100 ��g/mL streptomycim) (MEM+, control medium); (T2) MEM+IAA (40 ng/mL); (T3) MEM+IAA + epidermal growth factor (EGF) (100 ng/mL; Sigma, St. Louis, MO, USA); or (T4) MEM+EGF+FSH (100 ng/mL). After 6 days of culture of cortex tissue fragments in media, ultrastructural analysis was performed on preantral follicles (n = 3 in each treatment) included in a small cortical fragments. Preantral follicles were classified according to the stage of development as primordial follicles or as developing follicles. Preantral follicles cultured in supplemented media for 6 days were ultrastructurally normal. Their oocytes had an intact nucleus and cytoplasm that contained heterogeneous-sized lipid droplets, and numerous round or elongated mitochondria with intact parallel cristae were observed. Occasionally these organelles were associated with smooth endoplasmic reticulum (SER). Rough endoplasmic reticulum (RER) was rarely found. The cytoplasm of granulosa cells contained a large number of mitochondria and abundant RER. In contrast, follicles cultured in MEM+ (control) had a large number of vacuoles in the oocyte cytoplasm and excessive clustering of the chromatin material in the nucleus, suggesting an initial process of oocyte degeneration. In conclusion, the presence of IAA, EGF, FSH and their combinations helped to maintain ultrastructural integrity of ovine preantral follicles in cortical slices cultured in vitro.

Regulation of steady-state follistatin mRNA levels in rat granulosa cells in vitro

1992 ◽  
Vol 9 (2) ◽  
pp. 147-156 ◽  
Author(s):  
U. Michel ◽  
J. W. McMaster ◽  
J. K. Findlay
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Internal Standard ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Epidermal Growth

ABSTRACT The regulation of steady-state follistatin mRNA levels by different pituitary hormones and peptide factors was examined in granulosa cell cultures derived from diethylstilboestrol-treated immature rats. Cytosolic RNA from cell cultures was prepared by lysis and equal amounts of RNA from all samples were analysed with a solution—hybridization assay using a 32P-labelled antisense probe corresponding to a part of exon 5 together with a part of the 5′ end of exon 6 of the rat follistatin gene. In addition, a specific 35S-labelled probe for cyclophilin was used as an internal standard. The results show that 5 μg FSH/1 for 24 to 72 h stimulated steady-state follistatin mRNA levels, reaching levels 18·5-fold higher than controls. LH (0·2-100 μg/l) had only minor effects on follistatin mRNA levels in FSH-primed granulosa cells and prolactin, GH and IGF-I did not show any significant effects. Activin raised basal as well as FSH-stimulated steady-state follistatin mRNA levels up to ten- and twofold above controls respectively, whereas epidermal growth factor was found to inhibit FSH-stimulated follistatin mRNA levels in a dose-dependent manner. It is concluded that follistatin mRNA levels in granulosa cells are regulated by FSH rather than LH, and that the stimulation by FSH can be inhibited by epidermal growth factor but enhanced by activin. Activin alone was also capable of stimulating follistatin mRNA.

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