In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex

2018 ◽  
Vol 30 (2) ◽  
pp. 359 ◽  
Author(s):  
Érica S. S. Leal ◽  
Luis A. Vieira ◽  
Naíza A. R. Sá ◽  
Gerlane M. Silva ◽  
Franciele O. Lunardi ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Culture Media ◽  
Follicular Development ◽  
Vascular Endothelial ◽  
Fresh Tissue ◽  
Ovarian Cortex ◽  
Agnor Staining ◽  
Vitro Development ◽  
Endothelial Growth Factor

The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P < 0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P > 0.05). The average overall and daily follicular growth was highest (P < 0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P < 0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.

scholarly journals Insulin and Insulin-Like Growth Factor Stimulation of Vascular Endothelial Growth Factor Production by Luteinized Granulosa Cells: Comparison between Polycystic Ovarian Syndrome (PCOS) and Non-PCOS Women

2007 ◽  
Vol 92 (7) ◽  
pp. 2726-2733 ◽  
Author(s):  
Meghan B. Stanek ◽  
Sherri M. Borman ◽  
Theodore A. Molskness ◽  
Janine M. Larson ◽  
Richard L. Stouffer ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Polycystic Ovarian Syndrome ◽  
Culture Media ◽  
Vascular Endothelial ◽  
Vitro Fertilization ◽  
Endothelial Growth Factor ◽  
Ovarian Syndrome

Abstract Context: Vascular endothelial growth factor A (VEGF-A) is a potent cytokine that promotes angiogenesis and vascular permeability. After controlled ovarian stimulation (COS) for in vitro fertilization (IVF), excessive VEGF-A production can occur, particularly in women with polycystic ovarian syndrome (PCOS); however, it is unclear whether the regulation of VEGF-A production is different between PCOS and non-PCOS women. Objective: The aim of this study was to determine whether there were differences in the dose- and time-dependent effects of insulin and IGFs on VEGF-A production by luteinized granulosa cells (LGCs) from women with and without PCOS. Design and Setting: A prospective comparative experimental study was conducted at an institutional practice. Patients: Patients included six PCOS and six non-PCOS women undergoing COS and IVF. Interventions: Interventions included COS for IVF. Main Outcome Measures: VEGF-A levels in culture media were collected daily for 3 d from LGCs after incubation with variable doses of insulin, IGF-I, and IGF-II in the presence and absence of LH. Results: In both study groups, exposure to LH alone did not alter VEGF-A levels. However, insulin or IGF increased VEGF-A levels within 1 d and appeared to synergize with LH at 3 d. VEGF-A production by non-PCOS LGCs was more sensitive to IGF exposure, whereas PCOS cells were more sensitive to insulin. Although an increase in DNA content (P &lt; 0.05) was noted in cultures of PCOS cells, progesterone levels were lower compared with non-PCOS LGCs. Conclusion: Insulin and IGFs promote VEGF-A production in LGCs, but the response patterns are different when cells from PCOS and non-PCOS women are compared.

scholarly journals The Phytoestrogen Quercetin Impairs Steroidogenesis and Angiogenesis in Swine Granulosa Cells In Vitro

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Sujen Eleonora Santini ◽  
Giuseppina Basini ◽  
Simona Bussolati ◽  
Francesca Grasselli
Keyword(s):  
Granulosa Cell ◽  
Granulosa Cells ◽  
Follicular Development ◽  
Redox Status ◽  
Negative Influence ◽  
Animal Nutrition ◽  
Follicle Development ◽  
Vascular Endothelial ◽  
Endothelial Growth Factor

Experimental evidence documents that nutritional phytoestrogens may interact with reproductive functions but the exact mechanism of action is still controversial. Since quercetin is one of the main flavonoids in livestock nutrition, we evaluated its possible effects on cultured swine granulosa cell proliferation, steroidogenesis, and redox status. Moreover, since angiogenesis is essential for follicle development, the effect of the flavonoid on Vascular Endothelial Growth Factor output by granulosa cells was also taken into account. Our data evidence that quercetin does not affect granulosa cell growth while it inhibits progesterone production and modifies estradiol production in a dose-related manner. Additionally, the flavonoid interferes with the angiogenic process by inhibiting VEGF production as well as by altering redox status. Since steroidogenesis and angiogenesis are strictly involved in follicular development, these findings appear particularly relevant, pointing out a possible negative influence of quercetin on ovarian physiology. Therefore, the possible reproductive impact of the flavonoid should be carefully considered in animal nutrition.

scholarly journals Porcine ovarian cortex-derived putative stem cells can differentiate into endothelial cells in vitro

2021 ◽  
Author(s):  
Kamil Wartalski ◽  
Gabriela Gorczyca ◽  
Jerzy Wiater ◽  
Zbigniew Tabarowski ◽  
Małgorzata Duda
Keyword(s):  
Stem Cells ◽  
Vascular Endothelial Growth Factor ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Primary Component ◽  
Marker Genes ◽  
Vascular Endothelial ◽  
Ovarian Cortex ◽  
Endothelial Growth Factor

AbstractEndothelial cells (ECs), the primary component of the vasculature, play a crucial role in neovascularization. However, the number of endogenous ECs is inadequate for both experimental purposes and clinical applications. Porcine ovarian putative stem cells (poPSCs), although not pluripotent, are characterized by great plasticity. Therefore, this study aimed to investigate whether poPSCs have the potential to differentiate into cells of endothelial lineage. poPSCs were immunomagnetically isolated from postnatal pig ovaries based on the presence of SSEA-4 protein. Expression of mesenchymal stem cells (MSCs) markers after pre-culture, both at the level of mRNA: ITGB1, THY, and ENG and corresponding protein: CD29, CD90, and CD105 were significantly higher compared to the control ovarian cortex cells. To differentiate poPSCs into ECs, inducing medium containing vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor (EGF), ascorbic acid, and heparin was applied. After 14 days, poPSC differentiation into ECs was confirmed by immunofluorescence staining for vascular endothelial cadherin (VECad) and vascular endothelial growth factor receptor-2 (VEGFR-2). Semi-quantitative WB analysis of these proteins confirmed their high abundance. Additionally, qRT-PCR showed that mRNA expression of corresponding marker genes: CDH5, KDR was significantly higher compared with undifferentiated poPSCs. Finally, EC functional status was confirmed by the migration test that revealed that they were capable of positive chemotaxis, while tube formation assay demonstrated their ability to develop capillary networks. In conclusion, our results provided evidence that poPSCs may constitute the MSC population in the ovary and confirmed that they might be a potential source of ECs for tissue engineering.

scholarly journals Vascular endothelial growth factor production by human luteinized granulosa cells in vitro

1997 ◽  
Vol 12 (12) ◽  
pp. 2756-2761 ◽  
Author(s):  
A. Lee ◽  
L. K. Christenson ◽  
P. E. Patton ◽  
K. A. Burry ◽  
R. L. Stouffer
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Vascular Endothelial ◽  
Factor Production ◽  
Growth Factor Production ◽  
Endothelial Growth Factor

scholarly journals Follicle-Stimulating Hormone and Luteinizing Hormone/Chorionic Gonadotropin Stimulation of Vascular Endothelial Growth Factor Production by Macaque Granulosa Cells from Pre- and Periovulatory Follicles1

1997 ◽  
Vol 82 (7) ◽  
pp. 2135-2142
Author(s):  
Lane K. Christenson ◽  
Richard L. Stouffer
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Progesterone Production ◽  
Endothelial Growth Factor ◽  
In Vitro Treatment

Granulosa cells in the ovulatory follicle express messenger ribonucleic acid encoding vascular endothelial growth factor (VEGF), an agent that may mediate the neovascularization of the developing corpus luteum, but it is not known whether luteinizing granulosa cells synthesize and secrete VEGF during the periovulatory interval. Studies were designed to evaluate the effects of an in vivo gonadotropin surge on VEGF production by macaque granulosa cells (study 1) and to test the hypothesis that gonadotropins act directly on granulosa cells to regulate VEGF production (study 2). Monkeys received a regimen of exogenous gonadotropins to promote the development of multiple preovulatory follicles. Nonluteinized granulosa cells (i.e. preovulatory; NLGC) and luteinized granulosa cells (i.e. periovulatory; LGC) were aspirated from follicles before and 27 h after an ovulatory gonadotropin bolus, respectively. Cells were either incubated for 24 h in medium with or without 100 ng/mL hCG (study 1) or cultured for 6 days in medium with or without 100 ng/mL hCG or 0.1, 1, 10, and 100 ng/mL of recombinant human LH (r-hLH) or r-hFSH (study 2). Culture medium was assayed for VEGF and progesterone. In study 1, LGC produced 8-fold greater levels of VEGF than NLGC (899 ± 471 vs. 111 ± 26 pg/mL, mean ± sem; P &lt; 0.05). In vitro treatment with hCG increased (P &lt; 0.05) VEGF production by NLGC to levels that were not different from the LGC incubated under control conditions. In vivo bolus doses of r-hCG (100 and 1000 IU) and r-hFSH (2500 IU) were equally effective in elevating granulosa cell VEGF production. In study 2, in vitro treatment with r-hFSH, r-hLH, and hCG markedly increased (P&lt; 0.05) VEGF and progesterone production by the NLGC in a dose- and time-dependent manner. By comparison, the three gonadotropins (100 ng/mL dose) only modestly increased VEGF and progesterone production by LGC. These experiments demonstrate a novel role for the midcycle surge of gonadotropin (LH/CG or FSH) in primates to promote VEGF production by granulosa cells in the periovulatory follicle. Further, the data demonstrate that FSH-like as well as LH-like gonadotropins directly stimulate VEGF synthesis by granulosa cells.

scholarly journals In vitro and in vivo regulation of follicular formation and activation in cattle

2011 ◽  
Vol 23 (1) ◽  
pp. 15 ◽  
Author(s):  
Joanne E. Fortune ◽  
Ming Y. Yang ◽  
Wanzirai Muruvi
Keyword(s):  
Follicular Development ◽  
First Trimester ◽  
Vascular Endothelial ◽  
Second Trimester ◽  
Primordial Follicles ◽  
Kit Ligand ◽  
Secondary Stage ◽  
Endothelial Growth Factor

The establishment of a stockpile of non-growing, primordial follicles and its gradual depletion through activation of primordial follicles are essential processes for female fertility. However, the mechanisms that regulate follicle formation, the activation of primordial follicles to begin growth and the primary-to-secondary follicle transition are poorly understood, especially in domestic animals and primates. The authors’ laboratory is engaged in studying early stages of follicular development in cattle and this review summarises the progress to date. Bovine follicles begin to form in fetal ovaries around the beginning of the second trimester of pregnancy (about Day 90), but the first activated, primary follicles do not appear until after Day 140. Bovine fetal ovaries produce steroids and production is highest during the first trimester. In vitro, oestradiol and progesterone inhibit follicle formation and acquisition by newly formed follicles of the capacity to activate. Meiotic arrest of the oocyte in the diplotene stage of first prophase does not occur until after follicle formation and is correlated with acquisition of the capacity to activate. This may explain the gap between follicle formation and appearance of the first activated follicles. Once capacity to activate has been acquired, it seems likely that activation in vivo is controlled by the balance between stimulators and inhibitors of activation. Insulin and kit ligand stimulate and anti-Müllerian hormone (AMH) inhibits activation in vitro. Few bovine follicles transition from the primary to the secondary stage in vitro, but this transition is increased by medium supplements, testosterone and vascular endothelial growth factor (VEGF).

The distribution and expression of vascular endothelial growth factor A (VEGFA) during follicular development and atresia in the pig

2020 ◽  
Vol 32 (3) ◽  
pp. 259 ◽  
Author(s):  
Xiaomeng Gao ◽  
Jinbi Zhang ◽  
Zengxiang Pan ◽  
Qifa Li ◽  
Honglin Liu
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Granulosa Cells ◽  
Molecular Level ◽  
Follicular Development ◽  
Vascular Endothelial ◽  
Antral Follicles ◽  
Physiological Processes ◽  
Endothelial Growth Factor

The involvement of vascular endothelial growth factor A (VEGFA) in ovarian physiological processes has been widely reported, but the location and role of VEGFA during follicular atresia remain unknown. This study investigated the distribution and expression of VEGFA during porcine follicular development and atresia. Pig ovaries were obtained, individual medium-sized (3–5mm in diameter) antral follicles were separated and classified into healthy, early atretic or progressively atretic groups. Immunobiology and quantitative techniques were used to investigate the varied follicular distribution of VEGFA at both the morphological and molecular level. The results indicated that VEGFA protein expression peaked in tertiary follicles, mostly distributed in the thecal and inner granulosa layers, during follicular development while VEGFA mRNA was mainly expressed in the inner granulosa layers. Additionally, healthy antral follicles showed a significantly higher expression of VEGFA than atretic follicles in both theca and granulosa cells. Knockdown of VEGFA using siRNA revealed an antiapoptosis effect of VEGFA in cultured pig granulosa cells. Our results increase the knowledge of VEGFA functions in follicles.

Importance of vascular endothelial growth factor (VEGF) in ovarian physiology of mammals

Zygote ◽  
2011 ◽  
Vol 21 (3) ◽  
pp. 295-304 ◽  
Author(s):  
Valdevane Rocha Araújo ◽  
Ana Beatriz Graça Duarte ◽  
Jamily Bezerra Bruno ◽  
Cláudio Afonso Pinho Lopes ◽  
José Ricardo de Figueiredo
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Follicular Development ◽  
Vascular Endothelial ◽  
Follicular Cells ◽  
Ovarian Cells ◽  
Complex Process ◽  
Ovarian Folliculogenesis ◽  
Endothelial Growth Factor

SummaryOvarian folliculogenesis in mammals is a complex process. Several compounds have been tested during in vitro culture of follicular cells for a better understanding of the mechanisms and factors related to ovarian folliculogenesis in mammals. From these compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is strongly associated with angiogenesis and, in recent years, its presence in ovarian cells has been investigated extensively. Previous studies have shown that the presence of VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases during follicular development. Therefore, it is likely that the interaction between VEGF and VEGFR-2 is crucial to promote follicular development. However, few studies on the influence of this factor on follicular development have been reported. This review addresses aspects related to the structural characterization and mechanism of action of VEGF and its receptors, and their biological importance in the ovary of mammals.

Sign in / Sign up

Export Citation Format

Share Document