Biology and Medicine
Editor-in-Chief: Eytan R. Barnea MD, FACOG
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EARLY
PREGNANCY: Biology and Medicine Editor-in-Chief: Eytan R. Barnea MD, FACOG |
Objective
Basic characteristics of ovarian steroid production has largely been clarified by
the use of human granulosa cell cultures in vitro. This technique made also possible to
test different stimulatory and blocking hormones and chemicals in the cell cultures to
clarify regulatory mechanisms of the granulosa cells. However, these cell cultures are
static systems that do not give information about the dynamics of steroid production of
the granulosa cells. Therefore, in the present study we have tried to establish a dynamic
model using the so called superfusion method.
Methods
Granulosa cells were isolated from follicular fluid obtained by
aspiration from patients undergoing in vitro fertilization program. The granulosa cells
were packed into a closed Sephadex G10 column which was continuously kept at 37 °C and
washed with McCoy`s 5A culture medium. Samples of the medium leaving the column were
collected in every 30 minutes with a fraction collector. Stimulation with LH and blocking
with cycloheximide were carried out by adding the materials into the perfusing culture
medium. Progesterone levels of the samples were measured by RIA-s.
Results
We found that the basal progesterone secretion is pulsatile even in the absence of any
stimulation. After stimulation with LH there is a rapid, but a slight increase in the
steroid levels, followed by a delayed and also pulsatile definite increase of progesterone
levels starting at about 30 minutes.
The superfused cell column was introduced into endocrinological methodology by Lowry (1974) for studying the hypothalamo-pituitary-andrenocortical system. The method has been widely used to investigate the dynamics of the release of different pituitary hormone and the interaction between releasing factors and several brain peptides in a pituitary cell-superfusion system. This superfusion method is sensitive enough in demonstration of definite hormone responses even to low concentrations of stimulatory substances. Vigh and Schally 1984).
In the present study our main goal was to decide whether the superfusion system could also be used to test the dynamics of the hormonal secretion of hGC-s.
Materials and Methods
Preparation of the cells hGC were isolated from follicular fluid of patients underwent follicle aspirations in the course of IVF-ET. All patients were stimulated after GnRH analogue (Decapeptyl, Ferring, Kiel, Germany) long protocol desensitization with 2-6 ampoules injection of hMG (Humegon, Organon, Obeschleißheim, Germany) daily. Ovulation was induced with 10.000 IU of hCG 36 hours prior to the follicle aspirations. Follicles were needle-aspirated aided by ultrasound using a vaginal probe (Kretz 310 A). After the collection of oocytes the follicle fluid was centrifuged with 1000 x g for 10 minutes. The pelleted hGC mass was suspended in fresh McCoy's 5A medium (Sigma, St.Louis, MO, USA) supplemented with 25 mmol HEPES buffer (GIBCO Laboratories, Paisley Scotland). The obtained cell clusters were dispersed by repeated pipetting using Pasteur pipettes. hGC were separated from red blood cells by density gradient centrifugation for 20 minutes at 200-300 x g in 60% Percoll. After washing the cell pellet was resuspended in 1,0 ml McCoy's 5A medium supplemented with 0,35% bovine serum albumin (Sigma), 3 mmol L-glutamine (Gibco Laboratories), 105 IU/l penicillin and 100 mg/ml streptomycin sulfate (Seromed, Berlin, Germany). Viability of the cells was assessed by 0,5% Trypan-Blue (Serva, Heidelberg, Germany) exclusion (it regularly ranged between 80% and 95%). A small aliquot was used for counting the cells. 1x106 cells was mixed with 0,3 ml Sephadex G-10 which had been equilibrated with previously oxygenated medium. The mixture of hGC-s and Sephadex was transferred into the superfusion apparatus and perfused with the medium. The medium always freshly prepared and gassed continuously with a mixture of O2 (95%) and CO2 (5%) during the whole experiment. Superfusion apparatus The superfusion apparatus consisted of a number of 1 ml syringe barrels mounted vertically in a plexiglas holder which was kept at 37 °C by circulating water. Properly greased O-rings prevented the leakage of the circulating fluid into the syringe barrels. Each barrel was fitted with plungers at both ends. Holes were drilled in the plungers to accommodate plastic tubing. The lower plunger was covered with a small piece of 300 µm-pore nylon net to keep the Sephadex beads from escaping. The system was operated with a negative pressure, i.e. the flow through (5 ml/h) was collected with a multichanel peristaltic pump (Gilson Minipulse type HP-4) which was placed after the superfusion chamber(s). Test substances (LH, cycloheximide) were pumped into the system at 15 minute pulses after having established a stable baseline. The medium eluting from the chamber was collected at 30 minute intervals into disposable glass tubes (Bódis et al. 1993). Radioimuunoassays P was measured in the culture media samples using Coatria 125I-P RIA without extraction and dilution (bioMerieux, Marcy l'Etoile, France). The sensitivity of the assay was 0,15 nmol, the intra-and inter-assay coefficients of variation were in the higher range of the standard curve 4% and 4,4% respectively. Results Figure 1 shows the basal P secretion of hGC-s and after stimulation with different amounts of LH. It is obvious, that even the basal P secretion of hGC is pulsatile. A 15 minute long stimulus with 1µg of LH resulted in a rapid increase of the P level. The increment of P lasted about 1 hour, followed by a further 1 hour decrease of P levels to the baseline. With the same stimulation after 2 hours a pulsatile P secretion increase could be detected. 3 hours later a continuos stimulation with 1 µg/ml LH (5µg/h) was carried out, which resulted in a pulsatile, but in average a continuos increase in the P levels.To test the specificity of the results in the next experiment we used 2 parallel columns. One column was washed continuously with culture medium containing 2 µg/ml cycloheximide to block the P production of the hGC-s. As shown in Figure 2. in the presence of cycloheximide a continuos decrease of the basal P secretion could be detected. After stimulation with LH a slight increase of P was observed. In the other column after LH stimulation the pulsatile P increment could be detected. At 25 hours cycloheximide was added in the same concentration to the culture medium of this column. The result is a continuos decrease of P levels, even after LH stimulation.
Conclusions
The pulsatile secretion of P in the second half of the cycle is well known from the work of Filicori et al. However, this paper suggested, that pulsatility of the P is mere consequence of the pulsatile GnRH and LH release. From our data we conclude, that even the basal P secretion of hGC-s is pulsatile. The pulsatility of the P secretion is not due to the LH pulses, because a continuos stimulation with LH results in pulsatile P secretion.
Cycloheximide blocks the intracellular protein synthesis. Therefore it does not block the secretion of P stored in the hGC, but blocks the de novo synthesis of the hormone. From our experiments when cycloheximide was added into the culture medium we conclude, that the increase of P secretion after stimulation is mainly due to de novo synthesis of P. Using this dynamic system the conclusion could be drawn, that the P synthesis takes not longer than 60 minutes. That could also be an explanation of the 60 minutes pulses in the basal P secretion and after stimulation as well.
From these experiments we finally conclude, that the superfusion system could be used to assess the dynamics of the hGC’s hormone secretion. The system is suitable to test the effects of different hormones and drugs on hGC. Since the dynamics of the hormone secretion could be detected with the superfusion apparatus it provides more information than the static cell cultures.
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References
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