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R D Koos

Publications and source records attributed to R D Koos.

33 records · Page 2Linked to original sources
PubMed1987

Cyclic adenosine 3',5'-monophosphate-induced ovulation in the perfused rat ovary and its mediation by prostaglandins.

The role and mechanism of action of cyclic adenosine 3',5'-monophosphate (cAMP) in the ovulatory process was investigated by using the in vitro-perfused rat ovary model. Ovaries of pregnant mare's serum gonadotropin (PMSG, 20 IU)-primed rats were perfused for 21 h beginning in the morning of induced proestrus. In vitro stimulation with luteinizing hormone (LH; 0.1 micrograms/ml) resulted in 2.4 +/- 0.7 ovulations per treated ovary. Ovulations could also be induced by the addition of forskolin (30 microM) or dibutyryl cAMP (dbcAMP, 1 mM) with isobutylmethylxanthine (IBMX, 0.2 mM), with 11.8 +/- 1.9 and 18.6 +/- 4.4 ovulations per treated ovary, respectively. Indomethacin (5 micrograms/ml) significantly decreased the number of ovulations in the forskolin and dbcAMP + IBMX groups. The addition of prostaglandin E2 (PGE2; 1 micrograms/ml three times during the perfusion) to the forskolin + indomethacin group reversed the inhibition of ovulation (21.6 +/- 5.4 ovulations per treated ovary). Ovarian PGE tissue levels were significantly higher 10 h after stimulation with either LH, forskolin, or dbcAMP + IBMX compared to the unstimulated control group. Ovulated oocytes in the LH and forskolin groups resumed meiosis but oocytes in the dbcAMP + IBMX groups remained immature. This study shows that an increase in ovarian cAMP, even if not induced by LH, is sufficient to cause ovulation of preovulatory rat follicles, supporting the involvement of cAMP in the normal ovulatory process of the PMSG-treated rat. Furthermore, prostaglandin involvement in cAMP-induced ovulations is demonstrated.

Animals↗
PubMed1986

Stimulation of endothelial cell proliferation by rat granulosa cell-conditioned medium.

The ability of granulosa cells to produce mitogenic factors for vascular endothelial cells, factors which could potentially mediate angiogenesis in the ovary, was examined. Granulosa cells were obtained from preovulatory follicles of immature rats 48 h after priming with PMSG (20 IU). The cells (1 X 10(6)/well) were cultured in 3 ml serum-free medium 199 (M199) at 37 C without further treatment or in the presence of LH (100 ng/ml) or FSH (20 ng/ml). Since oxygen tension has been shown to regulate the production of angiogenic factors by other cell types, the cultures were carried out with either a high (20%) or a low (2%) oxygen concentration in the culture chamber. After 48 h, the medium was collected, filtered (0.2 micron), and frozen until tested for mitogenic effects on sparsely plated fetal bovine aortic endothelial cells. A 1:1 mixture of granulosa cell-conditioned M199 with fresh M199 plus 1% dialyzed fetal bovine serum resulted in 7- to 8-fold increases in endothelial cell numbers over the 4-day test period compared to controls (fresh M199 + 1% dialyzed fetal bovine serum only). Neither gonadotropin treatment nor the oxygen concentration during the conditioning period influenced the proliferation-stimulating activity of the medium. Medium conditioned by granulosa cells in 2% oxygen, however, did have an additional effect on endothelial cell morphology; the cells were more elongated and aligned than those treated with medium conditioned by granulosa cells in 20% oxygen, which showed a typical cobblestone morphology. Preliminary characterization studies indicate that both high (greater than 30,000) and low (less than 10,000) mol wt mitogenic factors are present. The mitogenic activity is heat resistant but partially destroyed by trypsin. The morphology-altering activity is confined to high mol wt fractions (greater than 30,000). These studies demonstrate that granulosa cells from preovulatory follicles release one or more factors in vitro which are mitogenic for endothelial cells. Furthermore, conditioned medium from granulosa cells cultured in low oxygen induces morphological changes in endothelial cells which suggest an increased propensity for migration.

Animals↗
PubMed1986

The regulation of granulosa cell proopiomelanocortin messenger ribonucleic acid by androgens and gonadotropins.

We have previously demonstrated the presence of proopiomelanocortin (POMC) messenger RNA (mRNA) in rat granulosa cells. This study examines the unique, tissue-specific regulation of granulosa cell POMC mRNA levels by hormones with established regulatory effects on these cells. Northern blot analysis of immature rat ovarian RNA revealed the presence of a single mRNA of approximately 900 base pairs in length. The levels of ovarian POMC mRNA increased approximately 6-fold 48 h after priming with PMSG when expressed per microgram of total RNA. Granulosa cells were isolated from immature PMSG-primed rats and cultured under serum-free conditions in the presence and absence of various hormones to determine their effect on POMC mRNA levels. Treatment of the cells for 48 h with LH (100 ng/ml), androstenedione (10(-7) M), or LH plus androstenedione elevated POMC mRNA levels. LH alone elicited a 5-fold increase in POMC mRNA, and androstenedione elicited a 10-fold increase; the combination treatment led to a 47-fold increase. The effects of the nonaromatizable androgen dihydrotestosterone (DHT) were also evaluated. Treatment for 48 h with DHT (10(-7) M) elicited a 40-fold increase in POMC mRNA levels. In vivo experiments measuring ovarian POMC mRNA from immature female rats corroborated the in vitro results. Animals injected sc with DHT (500 micrograms) demonstrated 5-fold increases in ovarian POMC mRNA when expressed per microgram of total RNA. These studies provide both in vitro and in vivo evidence that granulosa cell POMC mRNA is under unique hormonal regulation by both androgens and gonadotropins.

Androgens↗
PubMed1985

Comparison of the effect of 4-hydroxy-4-androstene-3,17-dione on aromatase 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.

Androstenedione↗
PubMed1985

Ovulations in rat ovaries perfused in vitro with follicle-stimulating hormone.

Using the model of the isolated perfused rat ovary, we have found that highly purified ovine follicle-stimulating hormone (FSH) preparations cause ovulation and that this effect is not due to luteinizing hormone (LH) contamination. Ovine FSH-13 at a concentration of 1.5 mU/ml induced ovulations in all perfused ovaries (8.8 +/- 2.3 ovulations/ovary), as did a more purified preparation, ovine FSH-211B, at concentrations of 0.5 mU/ml (15.0 +/- 6.4 ovulations/ovary) and 5 mU/ml (11.3 +/- 2.6 ovulations/ovary). This ovulation-inducing effect of FSH is accompanied by a marked stimulation of estradiol levels in the perfusion medium without stimulation of progesterone levels. Furthermore, a purified rat FSH preparation (15 mU/ml) also induced ovulation in all ovaries (13.8 +/- 2.2 ovulations/ovary) as well as a stimulation of both estradiol and progesterone in the medium. These data clearly confirm the direct ovulatory effect of FSH on the ovary.

Animals↗
PubMed1985

The effects of a gonadotropin-releasing hormone agonist on ovulation and steroidogenesis during perfusion of rabbit and rat ovaries in vitro.

The ability of a GnRH agonist (GnRHa) to exert direct effects on rat and rabbit ovaries was examined in vitro. Ovaries of estrous rabbits and immature, PMSG-primed rats were surgically removed and perfused with a defined medium via an aortic cannula. In this system, the ovary remains viable and capable of undergoing ovulation in response to LH. Samples of perfusion medium were taken for steroid measurements and the number of ovulations determined by direct observation (rabbit) or oocyte recovery (rat). Follicles of ovaries perfused with medium alone rarely ovulated. GnRHa (0.1 micrograms/ml) induced ovulations in 6 of 7 rat ovaries (4 to 22 ovulations per ovulating ovary) and this effect was blocked by a GnRH antagonist. In contrast, a much higher dose of the agonist (10 micrograms/ml) induced ovulations in only 7 of 15 rabbit ovaries. GnRHa caused small but significant increases in progesterone levels in the perfusion medium in both species in comparison to no treatment. Mean estradiol levels also tended to be higher in the GnRHa groups in comparison to controls but the differences were not significant. GnRHa appears to act directly on both the rabbit and rat ovary but the rat ovary is much more sensitive to its ovulation-inducing effects.

Animals↗
PubMed1984

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.

Androstenedione↗
PubMed1984

Perfusion of the rat ovary in vitro: methodology, induction of ovulation, and pattern of steroidogenesis.

A method for perfusion of the rat ovary in vitro for the study of ovulation was developed and characterized. Immature rats (27-29 days old) were primed with 20 IU of pregnant mare's serum gonadotropin (PMSG). Two days later a laparotomy was performed, the aorta and vena cava were cannulated, the right ovary was isolated, and all vessels connecting with the aorta and vena cava except for the right ovarian artery and vein were ligated. The preparation was placed in the perfusion apparatus and perfused for up to 20 h with oxygenated Medium 199 containing 4% bovine serum albumin (37 degrees C, pH 7.4). The perfusion pressure was 70-90 mm Hg and the average flow was 1 ml . min . ovary. Ovulation was confirmed by oocyte recovery from the apparatus. Nine ovaries were perfused without further treatment (controls); only 1 ovulation occurred in this group. Nine ovaries received luteinizing hormone (LH; 0.1 microgram/ml) 1 h after the start of perfusion; 71 ovulations resulted (range, 3-13/ovary). Ovulations did not begin before 9 h after LH administration. Samples of medium were taken frequently for measurement of progesterone, estradiol and androstenedione. Levels of all three steroids rose rapidly and markedly in response to LH but increased only slightly in control perfusions. This study demonstrates that ovulation in the rat ovary can be induced in vitro and provides a basis for further studies on the mechanism of ovulation.

Androstenedione↗
PubMed1983

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.

Animals↗
PubMed1982

Production of 6-keto-prostaglandin F1 alpha by rat granulosa cells in vitro.

The production of 6-keto-prostaglandin (PG)-F1 alpha by rat granulosa cells in vitro was measured in order to determine if the precursor of this compound, prostacyclin (PGI2), is a potential mediator of preovulatory changes in follicular function. Granulosa cells were collected from immature rats (27-29 days old) 48 h after an injection of PMSG (20 IU). The cells were incubated in medium 199 containing 1% BSA with or without arachidonic acid and various treatments for up to 5 h. PGI2 synthesis was determined by extracting the combined cells and medium, purifying the extract by thin layer chromatography, and measuring 6-keto-PGF1 alpha using a sensitive RIA. PGE synthesis was also determined by RIA in order to compare and contrast effects of treatments on PGE synthesis with those on 6-keto-PGF1 alpha synthesis. Both exogenous arachidonic acid and LH stimulated 6-keto-PGF1 alpha synthesis approximately 4-fold in comparison to controls during 5-h incubations. Maximum stimulation was achieved by the combination of arachidonic acid and LH. The effect of arachidonic acid was evident as early as 1 h of incubation, but LH had no effect until 3 h of incubation. PGE synthesis was also stimulated by arachidonic acid within 1 h of incubation and by LH within 3 h of incubation. A potent LHRH agonist also significantly stimulated 6-keto-PGF1 alpha and PGE production during a 5-h incubation, whereas three vasoactive agents (histamine, bradykinin, and angiotensin II) had no stimulatory effect on the synthesis of either compound. Based on the measurement of 6-keto-PGF1 alpha, it is concluded that rat granulosa cells have the capability to synthesize PGI2 and that this synthesis is stimulated by LH and a potent LHRH agonist. Therefore, PGI2 is a potential mediator of hormone actions in the preovulatory follicle.

6-Ketoprostaglandin F1 alpha↗
PubMed1977

Pregnant mare serum and human chorionic gonadotropin stimulate ovarian delta5-3beta-hydroxysteroid dehydrogenase in aged mice.

Aged (12- to 14-month-old) estrous and diestrous C57BL mice exhibited lower histochemically demonstrable ovarian delta5-3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity in thecal, luteal, and interstitial cells, and lower (P less than 0.01) ovarian 3beta-HSD concentration and total content than did young (3-month-old) estrous animals. Administration of pregnant mare serum (PMS, 10 IU subcutaneously), followed in 40 hours by human chorionic gonadotropin (HCG, 5 IU subcutaneously) or HCG (2 IU daily for 4 days) alone, restored luteal and interstitial 3beta-HSD in aged mice. Follicular, lutea, and interstitial 3beta-HSD activity was increased in aged mice by a single PMS injection (10 IU). The total ovarian dehydrogenase concentration was increased 100% in aged animals by PMS and/or HCG administration. Restoration of histochemically demonstrable ovarian 3beta-HSD and total enzyme content in aged mice by PMS and/or HCG indicates ovarian sensitivity to gonadotropin and subnormal tropic hormone stimulation of the ovary in situ.

Animals↗
PubMedDate not supplied

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.

1-Methyl-3-isobutylxanthine↗