Robert D. Koos

Inhibition of estrogen synthesis does not inhibit luteinizing hormone-induced ovulation

In isolated, perfused rat ovaries, the addition of luteinizing hormone (LH) to the perfusion medium consistently induced follicular ruptures. These ruptures were preceded by a marked increase in the levels of estradiol in the perfusion medium. The addition of 4-hydroxyandrostene-3,17-dione (4-OH-A) to the perfusion medium blocked this increase in estradiol, but did not prevent ovulation. Levels of estradiol in follicular fluid were also reduced during perfusion with LH plus 4-OH-A, in comparison to LH alone. The conclusion drawn was that the marked rise in the production of estrogen by preovulatory follicles of the perfused rat ovary, induced by an ovulatory dose of LH, is not required for ovulation to occur.

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused in vitro.

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused in vitro were measured in order to compare PG changes in this model system with those that occur in vivo and in isolated, LH-treated follicles in vitro. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 microgram/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17 beta. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement. Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other in vivo and in vitro models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.

The effect of reduced oxygen tension on progesterone accumulation in rat granulosa cell cultures

Studies were carried out on the effect of oxygen tension on progesterone (P) accumulation in rat granulosa cell cultures. At 1-2% oxygen, basal, luteinizing hormone (LH)-stimulated, and follicle stimulating hormone (FSH)-stimulated P accumulations were 20, 18, and 11%, respectively, of P levels at 20% oxygen. Basal P accumulation was also inhibited at 5% oxygen, but LH- and FSH-stimulated P levels were 50% and 40% higher, respectively, than at 20% oxygen. P levels at 10% oxygen were intermediate between those at 5% and 20% oxygen. The inhibitory effect of 1-2% oxygen on P accumulation was reversible: LH-stimulated P accumulation was inhibited in cultures incubated in 1-2% oxygen for 24 h, but rebounded during a subsequent 24 h period in 20% oxygen to the same level as that in cultures maintained continuously in 20% oxygen. We conclude that oxygen tension does influence granulosa cell steroidogenesis in vitro. Changes in blood flow and oxygen delivery to the ovary before and after ovulation could, therefore, effect the pattern of steroidogenesis during this period.

Ovulation in the perfused ovary in vitro: further evidence that estrogen is not required.

The effect of inhibition of estrogen synthesis on ovulation in rat ovaries perfused in vitro with medium without phenol red was examined. The addition of luteinizing hormone (LH, 0.1 microgram/mL) plus 3-isobutyl-1-methylxanthine (IBMX, 0.2 mM) to phenol red-free perfusion medium (M199 + 4% bovine serum albumin) induced ovulation. The number of ovulations was similar to that found in medium containing phenol red. There was a similar increase in estradiol (1, 3, 5 (10)-estratriene-3, 17 beta-diol) levels in the medium in both groups. The addition of 4-hydroxy-4-androstene-3, 17-dione (4-OH-A, 5 microM) to phenol red-free medium blocked the increase in estradiol levels induced by LH + IBMX, but did not prevent ovulation. There was no significant difference in the number of ovulations in the three groups. In conclusion, phenol red in the perfusion medium does not influence ovulation induced by LH + IBMX. Furthermore, an increase in estrogen is not required during the immediate preovulatory period for ovulation to occur.

Comparison of the effect of 4-hydroxy-4-androstene-3, 17-dione on aromatose activity in granulosa cells from preovulatory follicles of rats, rabbits, and humans

The effect of the aromatase inhibitor 4-hydroxy-4-androstene-3,17-dione (4-OH-A) on the synthesis of estradiol (1,3,5 (10)-estratriene-3,17 beta-diol) by granulosa cells from preovulatory follicles of rats, rabbits and humans was examined. Granulosa cells from all three species were incubated for 4 h without treatment (control) or in the presence of androstenedione (4-androstene-3,17-dione, 0.5 microM), 4-OH-A (5 microM), or both compounds together. Estradiol levels were determined in the medium and cells by radioimmunoassay. In all three species, estradiol synthesis was markedly increased by androstenedione and this increase was blocked by 4-OH-A. In the rabbit, however, 4-OH-A alone caused a small but significant increase in radioimmunoassayable estradiol. The apparent increase seen with 4-OH-A alone may be due to a metabolite of 4-OH-A that cross-reacts in the estradiol radioimmunoassay. With granulosa cells from humans, in which 4-OH-A is of potential therapeutic importance, no similar effect of 4-OH-A alone was observed.