Temporal desensitization of rat uteri for the decidual cell reaction is abolished by cholera toxin acting by a mechanism apparently not involving adenosine 3':5'-cyclic monophosphate

To determine if increased endometrial vascular permeability (a response which precedes decidualization) could be obtained in temporally nonsensitized uteri by treatments designed to increase endometrial adenosine 3':5'-cyclic monophosphate (cAMP) concentrations, cholera toxin (an activator of adenylate cyclase) was injected into the uterine lumen of immature rats treated to be at the equivalent of day 4, 5, or 6 of pseudopregnancy. In all experiments, the rats were pretreated with indomethacin to inhibit endogenous prostaglandin (PG) synthesis. Endometrial vascular permeability, determined using 125I-labeled bovine serum albumin, was assessed 8 h later. Cholera toxin increased endometrial vascular permeability to the same level in all groups. As determined by uterine weights 5 days after the intrauterine administration of cholera toxin or its vehicle, the toxin produced the same extent of decidualization in all groups. Cholera toxin had no detectable effect on uterine cAMP concentrations in rats sacrificed 15 min after intrauterine treatment. In contrast, intrauterine administration of PGE2 increased uterine cAMP concentrations at 15 min in all groups. These data suggest that the effects of cholera toxin and of PGE2 on endometrial vascular permeability and decidualization are not mediated by cAMP.     

Prostaglandin E2, adenosine 3':5'-cyclic monophosphate and changes in endometrial vascular permeability in rat uteri sensitized for the decidual cell reaction

The possibility that prostaglandin E2 (PGE2) increases endometrial vascular permeability and initiates decidualization in sensitized rat uteri by stimulation of adenosine 3':5'-cyclic monophosphate (cAMP) synthesis was investigated. Immature rats, pretreated so that they were sensitized for the decidual cell reaction, were used. Following the unilateral intrauterine injection of 50 microliters phosphate-buffered saline containing gelatin (PBS-G), a deciduogenic stimulus, uterine concentrations of both PGE and cAMP were elevated as early as 1 min after the intrauterine treatment. To determine if uterine stimuli which increase endometrial vascular permeability also increase uterine cAMP concentrations, rats, treated with or without indomethacin, an inhibitor of PG synthesis, received unilateral intrauterine injections of 50 microliters PBS-G with and without 10 micrograms PGE2 and were killed 15 min later. Uterine cAMP concentrations were elevated in all injected horns except in those of indomethacin-treated rats receiving PBS-G intraluminally, thus paralleling the expected changes in endometrial vascular permeability. As indicated by radioactivity levels in the stimulated horn 15 min after the i.v. injection of 125I-labeled bovine serum albumin, the intrauterine injection of dibutyryl cAMP, with or without theophylline, did not increase endometrial vascular permeability in indomethacin-treated animals. In contrast, cholera toxin, an activator of adenylate cyclase activity, markedly elevated permeability and induced decidualization. Except for the lack of a permeability response to the cAMP analogue, these data are consistent with the hypothesis that the effect of PGE2 on endometrial vascular permeability is mediated by cAMP.     

Estrogen induced changes in uterine sensitivity for the decidual cell reaction: Interactions between prostaglandin E2 and histamine or bradykinin

In rats receiving high doses of estrogen along with progesterone, the uterus is desensitized and does not respond to artificial stimuli with increased endometrial vascular permeability or decidualization. In addition, prostaglandin E₂ (PGE₂), the putative mediator of endometrial vascular permeability changes in sensitized uteri, is ineffective when given into the uterine lumen. The possibility that this inability of PGE₂ to increase endometrial vascular permeability may be related to the unavailability of hitamine of bradykinin was investigated. Rats were differentially sensitized for the decidual cell reaction by the daily injection of 2 mg progesterone with either 0.5 of 10 μg estrone for the 3 days preceding the unilateral intra-uterine injection of 50 μl phosphate buffered saline containing gelatin with or without 10 μg PGE₂ and with or without 1 mg histamine or 1 μg bradykinin. Prior to the intrauterine injection, all rats were treated with indomethacin to inhibit endogenous prostaglandin production. Endometrial vascular permeability changes were determined 8 h later by determining radioactivity levels in injected and non-injected uterine horns 15 min after the i.v. injection of ¹²⁵I-labelled bovine serum albumin. PGE₂ increased endometrial vascular permeability in rats receiving 0.5 μg estrone, but not in those receiving 10 μg. Histamine or bradykinin, alone or with PGE₂, did not affect endometrial vascular permeability in rats receiving either estrogen dose. The data suggest that the unresponsiveness of uteri from rats treated with high doses of estrogen is not simply due to the unavailability of bradykinin or histamine.     

Uterine decidualization in hypophysectomized-ovariectomized rats: effects of pituitary hormones

Decidualization of the endometrial stroma occurs in rats in response to implanting blastocysts or after the application of an appropriately timed artificial stimulus. It is well established that decidualization is regulated by estrogens and progesterone (P). The present study investigated the role of pituitary hormones in this response. Decidualization produced by the bilateral intrauterine injection of 100 microliter sesame oil was compared in ovariectomized (OVX) and hypophysectomized (HYPOX)-OVX rats. All animals were treated with a sequence of 17 beta-estradiol (E2) and P that in OVX rats supported decidualization. As assessed by uterine weights 5 days after uterine stimulation, decidualization was much greater in OVX than in HYPOX-OVX rats (geometric mean uterine weights of 1539 and 376 mg, respectively). To determine the ability of pituitary hormones to restore decidualization in HYPOX-OVX rats, animals were treated with ovine prolactin (oPRL, 2 x 100 micrograms daily), bovine growth hormone (bGH, 2 x 125 micrograms daily), and thyroxine (1 microgram/day, replacement for thyrotropin) in addition to E2 and P. Combined treatment with bGH + thyroxine resulted in decidualization which was not significantly different from that obtained in OVX rats; the effects of bGH and thyroxine were additive. oPRL had no significant effect. Administration of bGH + thyroxine during the prestimulation period resulted in decidualization which did not differ significantly from that obtained when the hormones were administered both pre- and poststimulation; administration during the poststimulation period only, when growth and differentiation of decidual cells occurs, resulted in much less decidualization. Because an increase in endometrial vascular permeability is a prerequiste for decidualization, [125I]-labeled bovine serum albumin was used to assess permeability 8 h after uterine stimulation. Uterine concentrations of radioactivity indicated that endometrial vascular permeability was increased to the same extent in bGH + thyroxine-treated HYPOX-OVX rats as in OVX animals; this increase was significantly reduced in vehicle-treated HYPOX-OVX rats. Because prostaglandins (PGs) are involved in decidualization, the possibility that the reduced responses in vehicle-treated HYPOX-OVX rats were a consequence of a decreased capacity of the uterus to produce PGs in response to the deciduogenic stimulus was investigated. As indicated by uterine PGE and PGF concentrations 15 min after uterine stimulation, uterine PGE and PGF production was increased by the stimulus in both vehicle-treated and bGH + thyroxine-treated HYPOX-OVX rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Evidence for the involvement of prostaglandins throughout the decidual cell reaction in the rat

Previous studies in which prostaglandin (PG) production was inhibited for a limited time by the s.c. administration of indomethacin have suggested that PGs are involved in the initiation of decidualization as well as the growth and differentiation of decidual cells. To reduce PG production during decidualization, in the present study indomethacin was infused from Alzet osmotic minipumps into the uterine lumen of ovariectomized rats with uteri sensitized for decidualization. To determine the effect of route of indomethacin administration on decidualization, rats received a single s.c. injection of indomethacin or its vehicle, and unilateral intrauterine infusion of indomethacin or its vehicle, in a factorial experiment. The inhibitory effects on decidualization, as assessed 5 days later by uterine weights, were greatest when both treatments were combined. Prostaglandins E and F concentrations 24 and 48 h after the insertion of the pumps were lower in the indomethacin-infused horns, suggesting that the indomethacin reduced uterine PG production. By contrast, subcutaneously administered indomethacin reduced uterine PG concentrations at 24 h but not at 48 h. Prostaglandin E2 and PGF2 alpha alone or combined, infused with indomethacin into the uterine lumen of rats treated subcutaneously with indomethacin, overrode the inhibitory effects of indomethacin. The dose-response relationships between these PGs and decidualization did not differ. These data suggest that PGs are required during the growth and differentiation of decidual cells from endometrial stromal cells.     

Stimulation of uterine ornithine decarboxylase in organ culture by decreasing osmolality: Possible relation to in vivo mechanisms

Ornithine decarboxylase (ODC) activity increases during many growth responses. Some cells and tissues in culture also exhibit elevated enzyme activity with decreasing osmolality of the culture medium. We have found that this also occurs with uterine tissue from ovariectomized rats. Organ culture incubation under hypotonic conditions caused maximal stimulation of uterine ODC activity at 4 hr. This stimulation was observed when either NaCl or sucrose was used to adjust the osmolality. Incubation under isotonic conditions also increased ODC activity relative to hypertonic conditions. This increase was similar in magnitude to that seen with unincubated uterine tissue from animals receiving systemic estradiol or intrauterine cholera toxin. Both estradiol and cholera toxin increase vascular permeability, and the resultant edema changes the extracellular microenvironment of the uterine cells. We suggest that this change somehow is mimicked by organ culture under hypotonic or isotonic conditions and is responsible for the stimulation of uterine ODC activity.     

Progesterone-induced meiosis in Xenopus laevis oocytes: a role for cAMP at the "maturation-promoting factor" level.

Cholera toxin inhibition of progesterone-induced meiosis of Xenopus laevis oocytes in vitro has been correlated with increased cAMP levels. Inhibition of germinal vesicle breakdown (Gvbd) and cAMP increase occurred after a lag period of 2 hr, when cholera toxin was injected, or 4--5 hr, when applied externally. The ability of the maturation-promoting factor (Mpf) to provoke Gvbd when injected into recipient oocytes was found to be dependent upon whether the oocytes had been exposed to cholera toxin alone or to toxin and progesterone. With the former, cAMP levels were elevated and Mpf activity was abolished, whereas with the latter, the increase in cAMP was less pronounced and Mpf activity was observed. Injection of cAMP or its 8-thio derivatives shortly before the appearance of progesterone-induced Mpf abolished Gvbd. If injected earlier or later, no inhibition was observed. In contrast, cholera toxin inhibited maturation even when added several hours before progesterone, suggesting a sustained accumulation of cAMP. No Gvbd occurred when 8-thio-methyl-cAMP was injected together with Mpf. These data suggest that cAMP is involved in the control of the formation/amplification and/or activity of Mpf-a result which may be of general significance in cell division mechanisms.     

Inhibition of 20 alpha-hydroxysteroid dehydrogenase activity by follicle-stimulating hormone and androgens in cultured rat granulosa cells: a search for the mechanism of action

Alterations of progesterone metabolism and especially of 20 alpha-hydroxysteroid dehydrogenase (20 alpha-HSD) activity were studied in cultured rat granulosa cells following various treatments. The cells were incubated for up to 48 h with or without follicle-stimulating hormone (FSH), androgens, hydroxyflutamide, estrogens, chlorea toxin, and dibutyryl cAMP [Bu2 cAMP]. Subsequently, the cells were incubated for 3 h with [4-14 C] progesterone (0.5 microM). The progesterone utilization and accumulation of 20 alpha-reduced and 5 alpha-reduced metabolites were assessed following thin-layer chromatography separation of radiolabeled steroids. Both FSH (1 microgram/ml) and testosterone (0.5 microM) decreased the 20 alpha-HSD activity by decreasing the maximal velocity (by 52% and 37%, respectively) without changing significantly the Km value. The inhibition of 20 alpha-HSD was demonstrable following 12 and 24 h exposure to FSH and following 24 and 48 h exposure to testosterone. Effects comparable to that induced by testosterone were elicited by other androgens (androstenedione and 5 alpha-dihydrotestosterone), but not by estrogens (estradiol-17 beta and estrone). Hydroxyflutamide reversed testosterone-induced effects: the increase of endogenous progesterone accumulation and the decrease of 20 alpha-HSD activity. Both cholera toxin (0.001-10 micrograms/ml) and Bu2 cAMP (62.5-1000 micrograms/ml) caused a dose-dependent inhibition of 20 alpha-HSD activity. Present results indicate that: the inhibition of 20 alpha-HSD by both FSH and androgens may be of a noncompetitive nature; androgen action on 20 alpha-HSD may be a true androgenic, receptor-mediated effect; and cAMP may mediate the FSH action on 20 alpha-HSD activity.     

cAMP-independent effects of cholera toxin on B cell activation. I. A possible role for cell surface ganglioside GM1 in B cell activation

Cholera toxin has been used as a tool to study the effects of cAMP on the activation of B cells but may have effects independent of its ability to elevate cAMP. We found five lines of evidence which suggested that cholera toxin suppressed mitogen-stimulated B cell activation through a cAMP-independent pathway. 1) Cholera toxin (1 microgram/ml) was consistently more suppressive than forskolin (100 microM) despite the induction of higher intracellular cAMP levels by forskolin. 2) Cholera toxin was more suppressive at 1 microgram/ml than at 0.1 microgram/ml despite equivalent elevations of cAMP. 3) Washing B cells following their incubation with cholera toxin reversed much of the inhibition without altering intracellular cAMP levels. 4) The A subunit of cholera toxin, which at high concentrations (10 micrograms/ml) induced levels of cAMP comparable to those induced by cholera toxin (1 and 0.1 microgram/ml), did not inhibit B cell activation. 5) cAMP derivatives at high concentrations were much less effective than was cholera toxin in suppressing B cell activation. Although the elevation of cAMP may cause a mild inhibition of B cell proliferation, we found that even a marked elevation of cAMP did not suppress B cell proliferation, unless the elevation was persistent. We did, however, observe that the degree of toxin inhibition more closely paralleled binding of the toxin to B cells than toxin stimulation of cAMP. This result raised the possibility that binding of cholera toxin to its ganglioside GM1 receptor mediated an inhibitory signal which suppressed B cell proliferation.     

Role of cyclic adenosine 3',5'-monophosphate in mediating the effect of prostaglandin E2 on decidualization in vitro.

When rat endometrial stromal cells from uteri sensitized for decidualization are cultured in vitro, there is an increase in alkaline phosphatase (ALP) activity paralleling that seen in vivo during decidualization. The addition of indomethacin to the culture medium decreases the endogenous production of prostaglandin E2 (PGE2) to below detectable levels and substantially reduces the increase in ALP activity. The addition of either PGE2 or its analog 16,16-dimethyl-PGE2, but not PGF2 alpha or its analog 15(S),15-methyl-PGF2 alpha, overrides this inhibitory effect, suggesting that PGE2 has a specific stimulatory effect upon ALP activity. This in vitro system was used to investigate the role of the cAMP pathway in mediating the stimulatory effect of PGE2 on ALP activity. The data indicate that PGE2 causes an increase in cAMP accumulation by the cells and that the addition of an analog of cAMP or substances which increase the level of cAMP in the cells (1-methyl-3-isobutyl xanthine, cholera toxin, forskolin) causes an increase in ALP activity. Collectively, the results suggest that the stimulatory effect of PGE2 is at least partially mediated by the cAMP pathway.     

6-Keto-prostaglandin E1 and the decidual cell reaction in rats

Although there is considerable evidence that prostaglandins (PGs) are involved in the decidual cell reaction in rats, which PGs are involved is uncertain. In the present study, we investigated the possibility that 6-keto-PGE1 is involved. To determine its ability to induce decidualization, 6-keto-PGE1 was infused unilaterally from Alzet osmotic minipumps into the uterine lumen of ovariectomized rats treated with estrogen and progesterone to sensitize their uteri for the decidual cell reaction. To reduce endogenous PG production, indomethacin was injected 2-3 h prior to pump insertion and was included in the vehicle for PG infusion. As determined by uterine weights 5 days after pump insertion, 6-keto-PGE1 and PGE2 produced decidualization which was equivalent. As indicated by a dose-response study, 6-keto-PGE1 and PGE2 did not differ in their ability to bring about decidualization. To determine if a deciduogenic stimulus resulted in increased uterine production of 6-keto-PGE1, as assessed by uterine concentrations, 6-keto-PGE1 and PGE concentrations in the uterus were determined after the unilateral intrauterine injection of 100 microliters sesame oil. There were no significant differences between stimulated and non-stimulated horns in 6-keto-PGE1 concentrations, whereas the concentrations of PGE2 were elevated in the stimulated horns. These data indicate that while both exogenous 6-keto-PGE1 and PGE2 induce decidualization, only uterine PGE concentrations are elevated by deciduogenic stimuli. Thus it is unlikely that 6-keto-PGE1 plays a role in decidualization.     

Cholera toxin stimulates division of 3T3 cells.

Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10(-10) M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.     

The sensitivity of the uterus of the mouse and rat to intraluminal instillation

The tissue accumulation of 125I-labelled human serum albumin 30 min after intravenous injection was used as an index of uterine vascular permeability. In ovariectomized mice, all sham and experimental instillation procedures produced a 6-10-fold increase in vascular permeability. Some effects were also manifest in the contralateral, control horn. In ovariectomized rats, instillation of saline and arachis oil increased vascular permeability 3-7-fold. After 3 or more days of progesterone treatment following oestradiol priming, fluorocarbon and arachis oil instillation produced marked vascular responses, but these were not restricted to the transient period in which the uterus would respond with decidualization. An IUD prevented the response to arachis oil instillation. These results indicate that the uterus is very sensitive to any manipulation and are consistent with decidualization representing a specialization of a normal uterine inflammatory response.     

Prostaglandin E2 enhances uterine stromal cell alkaline phosphatase activity in vitro

Prostaglandins have been implicated in the process of uterine decidualization in vitro, but sites of action are uncertain. Since one of the earliest changes in endometrial stroma following induction of decidualization is an increase in alkaline phosphatase activity, we have investigated the effects of PGs on stromal cell alkaline phosphatase activity in vitro. Immature rats were pretreated with hormones to sensitize their uteri for the decidual cell reaction. Endometrial stromal cells were isolated and cultured for up to 4 days with PGE2 (0-10 micrograms/ml) or PGF2 (0-10 micrograms/ml). Analysis of variance revealed a highly significant interaction between day of culture and concentration of PGE2 in medium (P less than 0.01). Stromal cell alkaline phosphatase activity decreased significantly with increasing culture duration (P less than 0.01). In the presence of PGE2, alkaline phosphatase activity was significantly higher (P less than 0.01) regardless of day of culture. In contrast, PGF2 alpha had only a small and inconsistent effect. These data indicate that PGs, and in particular PGE2, can act directly upon stromal cells.     

Induction of glucose-regulated protein 78 in rat uterine glandular epithelium during uterine sensitization for the decidual cell reaction

Endometrial receptivity for implantation and sensitization for decidualization in rodents is a transient state under the control of the ovarian steroids estrogen and progesterone. It is unclear, however, what molecular events mediate the onset of uterine receptivity. Messenger RNA differential display was performed on endometrial RNA from ovariectomized rats differentially sensitized for decidualization. Maximally sensitized uteri were at the equivalent of Day 5 of pseudopregnancy, and temporally nonsensitized uteri at Day 4 or 6; hormonally nonsensitized uteri were from animals on Day 5 treated with low or high doses of estradiol on Day 4. A cDNA with endometrial expression restricted to maximally sensitized uteri was isolated, cloned, and sequenced. The cDNA matched the sequence for glucose-regulated protein 78 (GRP78), a heat shock 70-related protein that resides in the lumen of the endoplasmic reticulum (ER) and has roles in several cellular processes including multimeric protein assembly, the degradation of proteins, and the storage and regulation of ER luminal calcium. Northern blot analysis indicated a dramatic increase in GRP78 mRNA levels restricted to the sensitized, Day 5 endometrium, suggesting a role in the onset of the sensitized phase. In situ hybridization and immunohistochemistry experiments localized the up-regulation of GRP78 within the receptive endometrium to the glandular epithelium.     

Effects of analogues of prostaglandin E2 and F2 alpha on the decidual cell reaction in the rat

The identity of the prostaglandins (PGs) involved in the decidual cell reaction is uncertain. In the present study we investigated the ability of analogues of PGE2 and PGF2 alpha, 16,16-dimethyl-prostaglandin E2, methyl ester (16,16Me2PGE2) and 15(S)-15-methyl-prostaglandin F2 alpha (15MePGF2 alpha) respectively, to bring about decidualization when infused into the uterine lumen of rats sensitized for the decidual cell reaction. As indicated by uterine weights 5 days after the commencement of the infusions into rats in which endogenous PG production had been inhibited by treatment with indomethacin, 16,16Me2PGE2 produced decidualization which was equivalent to that produced by PGE2. By contrast, the infusion of 15MePGF2 alpha inhibited decidualization, even when PGE2 was infused concomitantly. As indicated by uterine radioactivity concentrations after i.v. administration of 125I-labeled bovine serum albumin, the PGF2 alpha analogue also inhibited the endometrial vascular permeability increase which precedes decidualization. Compared to PGE2, 16,16Me2PGE2 was slightly less effective at displacing 3H-PGE2 from an endometrial membrane preparation; by contrast 15MePGF2 alpha was considerably less effective. These data suggest that PGE2 mediates the decidual cell reaction, and that the decidualization obtained in response to PGF2 alpha may involve its conversion within the uterus to PGE2.     

Evidence for a role for a uterine renin-angiotensin system in decidualization in rats.

Experiments were performed in vivo and in vitro to determine the effects of enalaprilat, a specific inhibitor of angiotensin-converting enzyme, on various aspects of the decidual cell reaction in rats. Ovariectomized, adult female rats were sensitized for the decidual cell reaction with steroid treatments. For in vivo experiments, intrauterine infusions of enalaprilat alone, and in combination with angiotensin II and prostaglandin E2 (PGE2), were initiated on the day of uterine sensitivity. Enalaprilat inhibited the increases in uterine PG concentrations, endometrial vascular permeability, alkaline phosphatase activity and uterine weight that occurred sequentially following infusion of vehicle. Concurrent infusion of angiotensin II did not reverse any of these inhibitory effects; PGE2 infusion partially, but not completely, reversed the inhibition of increase in uterine weight, although it did not alter the inhibition of endometrial vascular permeability. For in vitro experiments, endometrial stromal cells were obtained from uteri on the day of sensitivity and cultured for up to 3 days in the presence of enalaprilat and angiotensin II. Enalaprilat inhibited in a dose-dependent manner the increases in stromal cell alkaline phosphatase activity and media PGE concentration that occurred in the control cultures; these effects were fully reversed by concurrent treatment with angiotensin II. The inhibition of stromal alkaline phosphatase activity was also reversed by PGE2; conversely, the ability of angiotensin II to reverse the effect of enalaprilat was lost in the presence of indomethacin. These studies provide evidence of a requirement for angiotensin II during the decidual cell reaction in rats and suggest that it acts, at least in part, through a PG-dependent mechanism.     

Role of ovarian steroid hormones in the regulation of adenylate cyclase during early progestation

The hormonal regulation of uterine adenylate cyclase (AC) was measured in the rat by radiochemical analysis. Animals made pseudopregnant by cervical stimulation were ovariectomized on Day 1 (the first appearance of leukocytes in the vaginal smear) and injected for 6 days with sesame oil, 0.1-10.0 micrograms estrone, 2.0 mg progesterone, or 1.0 microgram estrone + 2.0 mg progesterone. AC activity in ovariectomized controls remained at basal levels (2.8-3.3 pmol cAMP formed/min X mg protein). The injection of progesterone did not alter AC activity significantly, but estrone increased AC activity during Days 3-5, and the response (5-17 pmol) was dose dependent. The action of estrone was not inhibited by progesterone. The present experiments revealed: a) AC from estrone-treated rats was activated 2- to 4-fold by 10 mM NaF; b) following treatment with estrone + progesterone, AC was activated 2- to 3-fold by a trauma to the uterus; c) unlike the response to fluoride, the effect of trauma was temporally limited to Day 4; and d) when AC was activated by trauma, no further increase was elicited by NaF. The data indicated that the transient sensitivity of AC to activation by trauma on Day 4 in E+P-treated rats was identical to that in intact rats and paralleled the normal timing of uterine sensitivity to decidual induction.     

Early effect of progesterone on levels of cyclic adenosine 3':5'-monophosphate in Xenopus oocytes.

Progesterone treatment of Xenopus oocytes in vitro causes progression through meiotic cell division. The role of altered intracellular levels of cAMP on the initiation of meiotic cell division has been studied. Basal levels of cAMP averaged 1.5 pmol in oocytes from eight females, and exposure to progesterone caused a rapid drop in cAMP to about 40 to 60% of basal. Half-maximal decreases occurred within 15 to 60 s, and cAMP returned to near basal values by 20 min after progesterone. Theophylline inhibition of progesterone-induced cell division was characterized by a small increase in basal levels of cAMP and a reduced drop in cAMP due to the hormone. Cholera toxin, an activator of adenylate cyclase, was found to be a potent inhibitor of progesterone-induced meiosis, with half-maximal inhibition at 8 times 10(-12) M. In addition, the purified A subunit of cholera toxin was an effective inhibitor of progesterone action when microinjected into oocytes, with half-maximal inhibition occurring at an approximate internal concentration of 1 X 10(-7) M. Cholera toxin alone increased cAMP levels by 20%, but upon addition of progesterone, the level increased transiently to 200% of basal, indicating that the inhibition was due to elevated levels of cAMP. The results support a model in which the initiation of meiotic cell division is regulated by cAMP and protein phosphorylation.