The physiological role of various neurotransmitters in the regulation of ovarian steroid secretion is still unclear. The possible role of catecholamines in the regulation of steroid secretion has been examined both in vitro and in vivo (1 - 3). The sympathic nervous system participates in the regulation of most of the organ systems, including the endocrine glands, although adrenergic effects on their hormone secretion are not well characterized. The physiological actions of catecholamines in the ovaries can be mediated by ß-adrenergic receptors, which are associated with the cAMP second messenger system (4 - 6). The primary catecholamines, adrenaline and noradrenaline, can interact with both a- and b-adrenergic receptors although their stimulatory effects on ovarian steroidogenesis in rat (7) cow (8) and sheep (5) can only be mediated by beta-receptors, since their response is inhibited by the beta adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine. The beta adrenergic receptor in swine ovarian tissue during the estrous cycle exhibited distinct qualitative and quantitative variations during different stages of the follicular and luteal development. The highest concentration of the specific beta adrenergic binding occurred in the corpus haemorrhagicum with a progressive decline in receptor content during the early and mid-stages of the luteal phase and a further loss of binding in the last phase of luteal function (9).

Therefore, it is possible that catecholamines may play a physiological role as luteotropic factors during luteal development or may be involved in the maintenance of luteal function during early pregnancy.

Numerous studies have demonstrated that loss of association with the oocyte affects the ability of granulosa cells to undergo proliferation (10 - 12). Several reports suggested the involvement of adrenergic nerves in ovarian function (4 - 6) Ovarian adrenergic nerves are localized principally around blood vessels and within stromal tissue of the ovary and their secretory products may participate in steroidogenesis (13). The physiological actions of catecholamines in the ovaries can be mediated by beta adrenergic receptors, which are associated with the cAMP second messenger system (4 - 7). The primary catecholamines, adrenaline and noradrenaline, can interact with both alpha and beta adrenergic receptors despite their stimulating effects on ovarian steroidogenesis in rats (7), cows (8) and sheep (5) and can only be mediated by beta-receptors, since their response is inhibited by the beta adrenergic antagonist propranolol, but not by the alpha-adrenergic antagonist phentolamine.

The growth, differentiation and steroidogenic capabilities of granulosa cells have most often been examined by culturing cells as monolayers in the presence of hormones and growth factors. However, in culture, granulosa cells lose the three-dimensional organization of the follicle, experience a disruption of their intercellular gap functional communication and undergo changes in cytosceletal structure due to lack of contact with the basal lamina (14).

This study was undertaken to investigate the influence of alpha- and ß-stimulators (alpha-stimulator: detomidinum HCL) as well as blockers (alpha-1 blocker: doxazosin, alpha-2 blocker: yohimbinum HCl, ß- blocker: carazolol) on bovine granulosa cells (GCs) culture from preovulatory follicles.


MATERIAL AND METHODS
Within 20 min. of slaughter, the ovaries were collected from all animals and placed in chilled medium TCM 199 modified with Earle’s salts, 25 mM HEPES (Sigma Chemical Company, St Louis, USA) and supplemented with 0,1 g/l L-glutamine, 0.1% BSA (w/v, fraction V, Sigma), 200 U/ml penicillin, 200 µg/ml streptomycin and 0.05 µg/ml amphotericin B (Gibco-BRL, Paisley, UK). The ovaries were then transported to the laboratory within 2 h of collection. Each ovarian follicle was classified as healthy or atretic on the basis of its morphological appearance (15). Granulosa cells were isolated from large follicles (>8 mm diameter) by gently washing the internal follicle wall with the medium described above containing sodium heparin (50 I.U. /ml, Polfa, Poland). Granulosa cells in the pooled washings were counted by using a hemocytometer and their viability was determined by the dye exclusion test (0.4 % trypan blue stain, Sigma). Before culturing the granulosa cells were centrifuged at 200g for 10 min, resuspended in a fresh medium devoid of sodium heparin and bovine serum albumin, but containing 10 % newborn calf serum (Gibco-BRL, Paisley, UK), 100 U/ml penicillin,100 µg/ml streptomycin and 0.025 µg/ml amphotericin B (Medium B) (15, 16), and recounted. Aliquots of approximately 4 x 105 viable granulosa cells in 0.5 ml medium were pipetted into individual wells of 4-well tissue culture plates (Nunc, Denmark) and cultured for 24 h at 37 °C in an atmosphere of 5 % CO2 and 95 % air. After 24 h of culture, the granulosa cells were examined under an inverted phase contrast microscope. Later the granulosa cell monolayers were washed twice and replaced in a fresh medium according to the following experimental design.

Granulosa cells were cultured for 120 hrs in Medium B containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of doxazosin, carazolol, detomidunum HCl and yohimbinum HCl). The control cells were cultured in Medium B without tested substances. Medium from the cultured cells was removed every 24 hrs and stored at -20°C until assayed for estradiol (E2), progesterone (P4) and testosterone (T4).

Estradiol, progesterone and testosterone assays

Estradiol, progesterone and testosterone were measured in unextracted granulosa cells culture medium by RIA. Estradiol recovery in the extracted samples was 88%. The sensivity of the assay for 17 ß estradiol was below 5 pg/ml, for progesterone below 0.15 ng/ml and below 0,1 ng/ml for testosterone. The intra- and inter-assay coefficient of variance was <9.0 %, and <10.2 % for estradiol, and <8.0 % and <11.1 % for progesterone and <5.6 % and <8.8 % for testosterone, respectively.

Data analysis

Statistical differences among the observed effects were determined by ANOVA. Data are presented as the mean ± SD of triplicate cultures. Two-tailed Student’s t-test was used to evaluate the significance of the difference between means of different studied concentrations. A p value < 0.05 was considered significant.


RESULTS
The results showed a decrease in P4 secretion after the addition of detomidinum HCl when compared with the control for all tested concentrations. Medium removed from the cultured cells with different concentrations (0.1, 1.0, 10.0 ng /ml) of detomidinum HCl didn’t show any statistic significant change in the concentration of P4 at 24 hrs of the culture (Figs 1, 2, 3). The highest level of P4 was measured in the medium with the concentration of detomidinum HCl of 0,1 ng/ml. We couldn’t find any statistical differences between the different concentrations of investigated alpha-2 AR agonist. In the medium recovered from the control cultured cells we have measured higher concentration of P4 in comparison with the tested substance in each concentration. The addition of carazolol at a concentration of 10.0 ng/ml caused a significant decrease in progesterone secretion after 48 hours of culture. Changes observed in other hormone levels did not differ statistically from the control. In the culture of granulosa cells with tested concentrations of carazolol in different concentrations we measured the increase of P4 for 72 hrs, after this time the concentration of P4 was higher in the control group (Figs 1, 2, 4).

Fig. 1. Concentrations of Progesterone (ng/ml) after 24 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Doxazosin, Carazolol, Detomidinum HCl and Yohimbinum HCl) (X+/-SD).

Fig. 2. Concentrations of Progesterone (ng/ml) after 48 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Doxazosin, Carazolol, Detomidinum HCl and Yohimbinum HCl) (X+/-SD).

Fig. 3. Concentrations of Progesterone (ng/ml) in the 120 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Detomidinum HCl) (X+/-SD).

Fig. 4. Concentrations of Progesterone (ng/ml) in the 120 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Carazolol ) (X+/-SD).

After yohimbinum addition a statistically significant decrease of progesterone was observed for all concentrations tested. After 48 hrs of culture this effect of yohimbinum wasn’t seen anymore. In the presence of doxazosin no differences in P4 activity was observed during the first 48 hrs and afterwards P4 couldn’t be measured in the medium. Doxazosin, when added to the culture medium, did not cause any statistically significant changes in P4. During the first 48 hrs of granulose cell culture with detomidinum HCl at a concentration of 0.1 ng/ml we found an increase of E2 concentration to more than 100 ng/ml medium. There was a significant difference between the test and control group. After 72 hrs, E2 couldn’t be measured any more after addition of detomidinum HCl at all concentrations (Figs 5, 6, 7). The concentration of E2 in the medium was lower (< 70 ng/ml) at 48 hrs than at 24 hrs (> 100 ng/ml).

Fig. 5. Concentrations of 17 ß Estradiol (ng/ml) after 24 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Doxazosin, Carazolol, Detomidinum HCl and Yohimbinum HCl) (X+/-SD).

Fig. 6. Concentrations of 17 ß Estradiol (ng/ml) after 48 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Doxazosin, Carazolol, Detomidinum HCl and Yohimbinum HCl)(X+/-SD).

Fig. 7. Concentrations of 17 ß Estradiol (ng/ml) in the 120 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Detomidinum HCl) (X+/-SD).

Fig. 8. Concentrations of 17 ß Estradiol (ng/ml) in the 120 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Carazolol ) (X+/-SD)

A slight testosterone (T4) increase was seen during the first 24 hours in the recovered medium of cultured granulosa cells after the addition of detomidinum HCl and carazolol in different concentrations. After 24 hrs T4 concentration didn’t change any more in the presence of tested substances at different concentrations. At 48 hrs high T activity was seen after adding detomidinum at a concentration of 0.10 ng/ml in the medium. When other concentrations of detomidinum were tested T levels remained always low. During 120 hrs of culture T activity was increased in the medium with detomidinum, especially in the concentration of 0.10 ng/ml. showed increasing activity. Until 48 hrs the increase was more or less the same and statistically significant (p<0,05). After 48 hrs of culture T could not be measured in the medium when doxazosin and yohumbinum HCl were tested (Figs 9, 10, 11, 12).

Fig. 9. Concentrations of Testosterone (ng/ml) after 24 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Doxazosin, Carazolol, Detomidinum HCl and Yohimbinum HCl) (X+/-SD).

Fig. 10. Concentrations of Testosterone (ng/ml) after 48 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Doxazosin, Carazolol, Detomidinum HCl and Yohimbinum HCl) X+/-SD).

Fig. 11. Concentrations of Testosterone (ng/ml) in the 120 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Detomidinum HCl) (X+/-SD).

Fig. 12. Concentrations of Testosterone (ng/ml) in the 120 hrs culture of Granulosa cells containing different concentrations (0.1, 1.0, 10.0, 100.0 ng/ml of Carazolol) (X+/-SD).

DISCUSSION
Results of our experiment demonstrate that amoung the drugs (detomidinum, doxazosin, yohimbinum and carazolol) used to promote the in vitro production of P4, E2 and T in granulosa cell culture, the alpha-2 agonist detomidinum decreased progesterone secretion independent of the given dosage when compared to the control group. The same results were observed when carazolol (ß-2 adrenoreceptors antagonist) was added to the culture medium. An increase of progesterone concentration was seen in the presence the alpha-1 adrenoreceptor blocker (doxazosin). The use of yohimbimum (alpha-2 adrenoreceptors antagonist) had no effect on progesterone production in the concentrations of 1.0 until 100.0 ng/ml. These results demonstrate that the catecholamines are able to influence progesterone production of granulosa cells in culture. In some publications (16 -21) it has been reported that the in vitro response of cultured granulosa cells to catecholamine treatment was only observed after several hours or even days. Our results showed a decrease in P4 secretion after the addition of detomidinum HCL when compared with the control for all tested concentrations. In contrast, in vivo reactions to the activation of the sympathic system are usually very rapid (17, 22, 23). The treatment with noradrenaline slightly decreased the progesterone production of FSH treated granulosa cells (25). The results suggest that catecholamines modulate the stimulating effect of gonadotropins. The level of noradrenaline present in corpora lutea are sufficient to modulate the production of progesterone by luteal cells in vitro (26). After yohimbinum addition a statistically significant decrease of progesterone was observed for all concentrations tested. E2 and T, however, did not significantly change when compared with the control. Doxazosin added to the culture medium did not cause any statistically significant changes in hormone secretion. The addition of carazolol (ß-AR blocker) in a concentration of 10.0 ng/ml caused a significant decrease in progesterone secretion only after 48 hours of culture. These results seem to support the hypothesis that drugs stimulating and blocking adrenergic receptors may play some role in ovarian steroidogenesis in cows. A slight testosterone increase was seen during the first 24 hours and then its concentration remained at a constant low level. It is well known that catecholamines are released during stress into the peripheral circulation exerting a negative influence on T biosynthesis (27, 28). It is interesting to point out that baseline levels of testosterone in the rats were significantly increased by the ß-2-receptor blocker (butoxamine), indicating that catecholamines may exert a tonic inhibitory effect on testosterone biosynthesis (28). Estradiol is known to exert a direct inhibitory effect on testosterone production by rat Leydig cells and it is possible that stress exerts at least part of its effects through such a mechanism (28, 30). Testosterone and catechol oestradiol have major effects on FSH-induced development of catecholamine responsive steroidogenesis whereas oestradiol mainly affects gonadotropin responsiveness in the granulosa cell culture system (31).

In vitro studies have shown that ß-adrenergic agents stimulate the increase of progesterone and cyclic AMP production in rat granulosa cells (32), swine granulosa and luteal cells (33) as well as bovine luteal cells (4, 20). Adrenaline added to bovine granulosa cells culture causes a rise in progesterone level (21). The stimulating effect of adrenergic agents on progesterone production in granulosa cells exposed to FSH activity is probably caused by stimulation of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and inhibition of 20 alpha hydroxysteroid dehydrogenase (20 alpha HSD) (34). Noradrenaline and adrenaline increase the androgen production stimulated by LH in external theca cells and penetrate through the basement membrane of theca and together with PRL, FSH and LH may stimulate progesterone production in granulosa cells (34). In our investigation a slight increase in 17ß estradiol secretion was also observed. Noradrenaline and adrenaline can also indirectly influence the estradiol production in granulosa cells through a rise in the concentration of androgens produced in internal theca cells (35). The increased production of progesterone after adding the granulosa to the cell culture medium suggests an involvement of catecholamine in the regulation of the ovarian follicles secretory function (21). The gonadotrophins used to promote follicular development in vivo leads to differences in granulosa cell steroidogenesis which are evident after luteinization and culture. This additionally supports the notion that the environment of follicular development will be reflected in the resulting of corpus luteum activity (36).


CONCLUSION
Our data confirm that different neurotransmitters have a direct action on the steroid production of cows granulosa cells in vitro. Progesteron and 17b estradiol secretion by cows granulosa cells can be affected by Detomidinum HCI inducing a decrease of progesterone and an increase of 17ß estradiol secretion by granulosa cells in vitro can be inhibited by Yohimbinum HCI as a specific alpha-2 adrenergic receptor blockade. Carazolol has an inhibitory effect on progesterone secretion by granulosa cells in vitro. The specific alpha-1-adrenergic receptor antagonist Doxazosin had no influence on secretion of P4, E2 and T4, in granulosa cells in vitro.


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