No abstract

Adenosine has been shown to acutely amplify LH-dependent events in luteal cells and FSH-dependent events in granulosa cells. In this study, the specificity of purines on mature rat granulosa cell ATP levels in short term culture was assessed, and a comparison of the relative effect of adenosine on amplification of FSH- and LH-stimulated cAMP accumulation was made. Adenosine rapidly and significantly increased ATP levels in granulosa cells. This effect was maximal (approximately 2-fold) within 60 min of culture and occurred in the absence or presence of FSH or LH. The increase in granulosa cell ATP levels by adenosine was dose dependent, with half-maximal and maximum responses of 10 and 30 microM adenosine, respectively. Dipyridamole (10 microM), a purine transport inhibitor, blocked the adenosine-dependent increase in granulosa cell ATP levels. Adenosine and 5'-AMP were equipotent in increasing cell ATP levels; adenine also increased ATP levels, but was significantly less active (approximately 50% of adenosine), whereas hypoxanthine, inosine, and xanthine were inactive. FSH was consistently found to decrease granulosa cell ATP levels by about 30% in the absence or presence of adenosine, whereas LH had no effect on cell levels of ATP. Both FSH and LH significantly stimulated cAMP accumulation in granulosa cells, but the maximal response to FSH was substantially greater than that to LH. Adenosine significantly amplified cAMP accumulation in response to both FSH and LH, but the effect of adenosine on this response to FSH was modest. Amplification by adenosine of cAMP accumulation in response to LH was substantial and about 2- to 3-fold greater than that seen with FSH. These studies show that purines acutely and specifically increase ATP levels in rat granulosa cells. Since adenosine augments LH-dependent cAMP accumulation to a greater extent than FSH-stimulated cAMP production, we suggest that adenosine may favor premature follicular luteinization and, perhaps, function as a mediator of atresia in the developing follicle.

No abstract

The objective of the present studies was to examine adenosine uptake in the rat luteal cell, to characterize the cellular products after uptake, and to assess the role of adenosine transport and conversion to cAMP in amplification of LH-stimulated cAMP accumulation. Adenosine uptake showed an apparent Km of 7.3 +/- 0.6 microM, and a maximum velocity of 2.2 +/- 1.4 pmol/min X 10(5) cells at 24 C; uptake was temperature dependent (Q10 = approximately 3) and inhibited by dipyridamole (IC50 = 7 microM). Radiolabeled adenosine uptake was inhibited by AMP (IC50 = 14 microM), ATP (IC50 = 16 microM), guanosine (IC50 = 20 microM), inosine (IC50 = 22 microM), ADP (IC50 = 26 microM), and theophylline (IC50 = 5 mM); no inhibition by adenine, hypoxanthine, xanthine, prostaglandin F2 alpha (PGF2 alpha), PGE2, or LH was seen. Cellular products of radiolabeled adenosine uptake were found primarily in the trichloroacetic acid-soluble fraction (88%), and 90% of the radioactivity in this fraction comigrated with adenine nucleotides on electrophoresis; time-dependent incorporation of radioactivity into RNA, DNA, and protein was also seen. Adenosine transport did not appear to be related to the functional state of the luteal cell; for example, no change in the characteristics of uptake was seen in cells obtained from hypophysectomized animals or in cells incubated directly with PGF2 alpha or LH. Adenosine increased cell ATP levels in a dose-dependent manner in parallel with amplification of LH-stimulated cAMP accumulation. A substantial proportion of the total cAMP produced by the cells was derived from extracellular adenosine (40-90%). This response was directly related to the concentration of adenosine, and LH increased the magnitude of cAMP derived from adenosine by about 2-fold. Based on the present studies, adenosine uptake in the luteal cell appears to occur by a dipyridamole-sensitive, phosphorylation-dependent transport system which is independent of pituitary hormones or PG regulation. Moreover, amplification of LH-dependent cAMP accumulation by adenosine appears to be primarily a mass effect due chiefly to utilization of extracellular adenosine by the cell as a prosubstrate for conversion into cAMP by adenylate cyclase.

The steroidogenic capability of granulosa cells isolated from 12 preovulatory human follicles was correlated with the stage of maturation of the corresponding oocyte-corona-cumulus-complex ( OCCC ). Individual follicles from human menopausal gonadotropin (hMG) stimulated cycles were aspirated 36 h after administration of hCG. Granulosa cells were cultured for 150 min and corresponding OCCC were evaluated for maturity before fertilization with human sperm. Granulosa cell aromatase activity was measured using 1 beta-3H-testosterone as substrate by quantitating the amount of 3H2O produced. Progesterone production by the granulosa cells was measured as was follicular fluid levels of combined hCG and LH activity and FSH and PRL. Follicular fluid concentrations of combined hCG plus LH activity decreased somewhat while FSH levels increased as OCCC matured. PRL levels did not vary. Granulosa cell progesterone production did not change with maturity of OCCC . However, aromatase activity decreased as OCCC matured with levels from granulosa cells with immature OCCC vs. intermediate and mature OCCC of 260 +/- 148 vs. 129 +/- 53 (SE) pg E2/10(5) cells, respectively (P less than 0.07). Although granulosa cells responded variably to hMG stimulation from individual to individual, and the response was not predictable from peripheral serum estradiol levels, follicles isolated from the same patient had a definite diminution in aromatase activity with OCCC maturation. From these preliminary results, aromatase activity in immediately preovulatory granulosa cells declined as OCCC matured in hMG/hCG stimulated cycles.