Estimation of inhibin-like activity in spent medium from rat ovarian granulosa cells during long-term culture
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Antral follicle
Granulosa cell
Follicular fluid
In vivo, follicular fluid bathes the granulosa cells and cumulus-oocytecomplex. Not surprisingly, components of follicular fluid can promote oocyte development; however, the exact mechanism by which the fluid is beneficial remains unknown. The content of follicular fluid varies dynamically during antral folliculogenesis, and its use in modeling culture medium formulation has been recognized, albeit not fully explored. Oxidative stress arises whenever there is an imbalance in proand anti-oxidants. In this respect, it is conceivable that follicular fluid assists in controlling levels of pro-oxidants within the follicle. Therefore, whether follicular fluid directly provides protection of follicle cells from oxidative damage remains to be ascertained. This study focuses on the profiling of follicular fluid for the central antioxidant superoxide dismutase (SOD) and its three isoforms (CuZn-SOD, Mn-SOD, and extracellular or EC-SOD). Given documented and predicted changes in metabolic requirements and oxygen tension during folliculogenesis, we hypothesized that a dynamic balance of antioxidants (specifically SODs) characterizes the follicular fluid of developing antral follicles. To test our hypothesis, follicular fluids were collected from antral follicles that were sized following dissection from bovine ovaries. Ovaries were obtained at random stages of follicular development and the estrous cycle. Each follicular fluid sample was subsequently analyzed for: (1) the total levels of SOD enzymatic activity, and (2) the expression and quantification of Cu, Zn-SOD, Mn-SOD, and EC-SOD by immunoblotting. Amounts of SOD protein and activity were then evaluated in accordance with: (1) the size of the antral follicle that the follicular fluid originated from (specifically at times when the developmental competency of oocytes is known to vary); and (2) recorded ovarian parameters (stages of follicular development and the estrous cycle). Our work contributes to the identification of novel factors that relates to oxidative stress and that may influence the normal function of granulosa cells and cumulus-oocyte-complexes. In future studies, efforts aimed at optimizing culture conditions will benefit from such knowledge of the natural environment within which oocytes develop. (poster)
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Follicular fluid
Atrial natriuretic peptide
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The effect of follicular fluid on FSH stimulated progesterone secretion and LH/hCG receptor induction was studied in granulosa cells harvested from small porcine follicles. The stability of the stimulatory factor was also assessed. Follicular fluid from large porcine follicles (LFF) added to the culture of granulosa cells in the dilution from 1:32 to 1:3 stimulated progesterone secretion, while at higher concentrations (dilution 1:2 and 1:1) it had an inhibitory effect. In contrast, the influence of follicular fluid on the induction of LH/hCG receptor was stimulatory only in the concentration of LFF diluted from 1:3 to 1:1. When the granulosa cells were cultured with follicular fluid in the absence of pig serum the effect on both progesterone secretion and hCG binding was stimulatory in all the concentrations of follicular fluid tested. Different treatment of follicular fluid (charcoal extraction temperature treatment, lyophilization, dialysis, ethanol precipitation, delipidation and ether extraction) showed that the stimulator of granulosa cell luteinization was not a stable compound.
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Reproductive toxicity
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Endocrine and gametogenic functions of the ovulatory follicle may be linked. To verify this, we studied granulosa cell steroidogenesis in relation to oocyte fertilization and preimplantation embryo development in vitro. Multiple follicles were stimulated in in vitro fertilization patients with clomiphene citrate and ovulation was induced with human chorionic gonadotropin (hCG). Oocytes were fertilized with husband’s sperm and normal embryos were replaced 48 h later. Granulosa cells were separated from follicular fluid from 64 follicles and incubated for 3 h with and without aromatase substrate (1 µM testosterone). Progesterone and estradiol levels were measured in follicular fluid and incubation medium. Follicular fluid steroid levels and granulosa cell steroidogenesis showed no significant differences for oocytes which cleaved normally and those which did not. Granulosa cell aromatase activity was high in all follicles, suggesting that the low periovulatory follicular fluid estradiol level is not explained by a fall in granulosa cell aromatase after hCG. High granulosa cell progesterone production and follicular fluid progesterone were consistent with advanced granulosa cell luteinization. Oocytes undergoing polyspermic activation were from larger follicles with elevated follicular fluid progesterone levels, suggesting that follicular size and follicular fluid progesterone are correlated with “over-ripeness” and polyspermy. No simple relationship exists between oocyte function and the present indices of granulosa cell steroid metabolism.
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Despite having amenorrhea and markedly elevated serum gonadotropin levels, some women with karyotypically normal spontaneous premature ovarian failure, nevertheless, have ovarian follicles that function intermittently. Graafian follicles capable of responding to these high FSH levels are faced with high serum LH levels as well, which might induce inappropriate luteinization and prevent normal follicle function. We examined this possibility using weekly blood sampling and sonography in 65 patients. Nearly 50% of our patients demonstrated ovarian follicle function [serum estradiol, > 183 pmol/L (50 pg/mL)] during a median of 4 months of observation (range, 2-6 months). However, during this observation, only 16% achieved an ovulatory serum progesterone level [> 9.5 nmol/L (3.0 ng/mL)]. We imaged an antral follicle by sonography in over 40% of patients (27 of 65), and serum estradiol was significantly greater when an antral follicle was present. The follicles in these patients were not functioning normally, however. In contrast to normal women, patients with ovarian failure had poor correlation between follicle diameter and serum estradiol. We biopsied these antral follicles in 6 patients and found luteinized Graafian follicles in all cases. Therefore, luteinized Graafian follicles account for at least 60% of the antral structures imaged (95% confidence limit). Thus, inappropriate luteinization of Graafian follicles appears to be a major pathophysiological mechanism in patients with karyotypically normal spontaneous premature ovarian failure.
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In ovaries surgically removed for fertility preservation, hormone concentrations in fluid from small antral follicles were determined. Levels were compared with those found in preovulatory follicular fluid.The objective of this study is to measure intrafollicular concentrations of anti-Müllerian hormone (AMH), inhibin-A, inhibin-B, estradiol, and progesterone.The study was set in a university hospital.Patients were 22 women suffering from a cancer disease and 16 women undergoing assisted reproduction.Fluid from 35 follicles (diameter, 3-8 mm) was included and compared with that of 32 preovulatory follicles.The main outcome measures were intrafollicular concentrations of the measured hormones and their possible correlation.Concentrations of AMH in small antral follicles were almost three orders of magnitude higher than in follicle fluid of preovulatory follicles, 790 +/- 95 vs. 1.17 +/- 0.14 ng/ml (mean +/- sem), respectively. There was a significant negative correlation between estradiol and AMH in fluid from small antral follicles, whereas inhibin-A and inhibin-B were correlated positively with estradiol concentrations. Progesterone showed a similar correlation to levels of AMH but only in fluid of preovulatory follicles.The high expression of AMH in granulosa cells of small antral follicles actually translates into very high follicle fluid AMH concentrations. This most likely explains the correlation between serum AMH levels and the number of small antral follicles as previously demonstrated. The negative correlation between estradiol and AMH suggests that FSH down-regulates AMH expression. Thus, the microenvironment of the follicle shows profound changes with developmental stage and highlights the importance of studies to understand the mechanisms that regulate follicular growth and development during antral stages of development.
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Anti-Müllerian hormone
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Follicular fluid
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Antral follicle size might be a valuable additive predictive marker for IVF outcome. However, while some studies show positive relations between follicle size and reproductive outcome, others have not been successful in establishing this relation. To better understand consequences of antral follicle size for reproductive outcome, we aimed to obtain insight in follicle size-related granulosa cell processes, as granulosa cells play an essential role in follicular development via the production of growth factors, steroids and metabolic intermediates, needed for follicular growth and oocyte development. Using pigs as a model, we compared gene and protein expression in granulosa cells of smaller and larger follicles in the healthy antral follicle pool at the start of the follicular phase of the estrous cycle. In sows, the early antral follicle pool is very heterogeneous when e.g. size and steroid content of the follicular fluid are considered. To which extent this variety contributes to the developmental competence of the follicles is not clear. Therefore, sows with high variation in antral follicle size (HighVAR) as well as sows with low variation in antral follicle size (LowVAR) were used. Granulosa cells of smaller antral follicles in the healthy antral follicle pool show increased cell proliferation, which was accompanied by a metabolic shift towards aerobic glycolysis (i.e. the Warburg effect), similar to other highly proliferating cells. High granulosa cell proliferation rates in smaller follicles might be regulated via increased granulosa cell expression of AR and EGFR which are activated in response to locally produced mitogens. While granulosa cells of smaller follicles in the pool were more proliferative, indicative of higher follicular growth, granulosa cells of larger follicles in the pool showed less proliferation and were more differentiated, as they showed a higher expression of follicular maturation marker IGF1 and ANGPT1. Our results imply that the inclusion of strict criteria of antral follicle size in IVF protocols might improve reproductive outcome. In addition, we have granulosa cell gene expression of healthy follicles to unravel underlying mechanisms of differences in COC morphology. We compared gene expression in sows with low vs. high-COC-health and found a decreased expression of genes involved in ovarian steroidogenesis (e.g. CYP19A1, ADM, SPP1) and higher expression of genes involved in follicular atresia (e.g. GADD45A, INHBB) in sows with low-COC-health. Thereby we have identified several genes which may serve as markers for follicle developmental competence.
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