Stimulation by cyclic nucleotides of prostaglandin E production in isolated graafian follicles.

Rat Graafian follicles isolated intact responded to 8-Br-cyclic AMP and 8-Br-cyclic GMP with increased prostaglandin E (PGE) production during a 6 h incubation. By contrast, 8-Br-cyclic IMP, 8-Br-5' AMP and 8-Br-5' GMP were inactive in this respect. The effect of 8-Br-cyclic AMP and 8-Br-cyclic GMP was noted only after a lag period of about 4 h. Choleragen, LH, and the phosphodiesterase inhibitor (3-isobutyl-l-methyl-xanthine; IBMX) also stimulated PGE production. Actinomycin D and cycloheximide given simultaneously with 8-Br-cyclic AMP or LH prevented the stimulatory effect of these agents. Concomitant addition of arachidonic acid did not overcome the effect of these inhibitors. Administration of hCG in vivo or incubation with LH in vitro did not elevate endogenous ovarian free arachidonate, while PGE production was enhanced. Dexamethasone prevented this stimulatory effect of hCG. Collectively, the results suggest that stimulation of ovarian PGE production by cyclic nucleotides and LH is dependent on de novo synthesis of one or more components of the PG synthetase system rather than on substrate availability. Cyclic nucleotides may mediate the stimulatory effect of gonadotropins on PGE production.     

The need of protein synthesis for the effect of LH--RH on the cAMP level in the anterior pituitary in vitro

The effect of LH--RH on cAMP accumulation in the anterior pituitary of male rats was investigated. The effect consisted of two phases. In the first phase of incubation (about 2 hours), there was no change in the cAMP level, while in the second phase a significant increase was observed. Cycloheximide (10(-5)M) given simultaneously with the LH--RH totally blocked the observed effect of LH--RH. If cycloheximide was given in the second phase of incubation, when the cAMP level was already elevated, its further rise was prevented and the elevated cAMP level remained unchanged for 2 or more hours. Actinomycin D (10(-5)M) added together with LH--RH totally abolished the action of the latter, but if actinomycin D was given 1 hour after LH--RH a significant increase of the cAMP level was found at the end of the 4-hour incubation.     

Stimulation by cyclic nucleotides of prostaglandin E production in isolated Graafian follicles

Rat Graafian follicles isolated intact responded to 8-Br-cyclic AMP and 8-Br-cyclic GMP with increased prostaglandin E (PGE) production during a 6 h incubation. By contrast, 8-Br-cyclic IMP, 8-Br-5′ AMP and 8-Br-5′ GMP were inactive in this respect. The effect of 8-Br-cyclic AMP and 8-Br-cyclic GMP was noted only after a lag period of about 4 h. Choleragen, LH, and the phosphodiesterase inhibitor (3-isobutyl-1-methyl-xanthine; IBMX) also stimulated PGE production. Actinomycin D and cycloheximide given simultaneously with 8-Br-cyclic AMP or LH prevented the stimulatory effect of these agents. Concomitant addition of arachidonic acid did not overcome the effect of these inhibitors.Administration of hCG $\text{in vivo}$ or incubation with LH $\text{in vitro}$ did not elevate endogenous ovarian free arachidonate, while PGE production was enhanced. Dexamethasone prevented this stimulatory effect of hCG.Collectively, the results suggest that stimulation of ovarian PGE production by cyclic mucleotides and LH is dependent on $\text{de novo}$ synthesis of one more components of the PG synthetase systme rather than on substrate availability. Cyclic nucleotides may mediate the stimulatory effect of gonadotropins on PGE production     

Formation of cyclic adenosine monophosphate (cAMP) in the preovulatory rabbit follicle: role of prostaglandins and steroids

The preovulatory increase in follicular prostaglandins (PG) stimulated by luteinizing hormone (LH) is dependent upon 3'-5'-cyclic adenosine monophosphate (cAMP) and is essential for ovulation. It has been proposed that follicular PG stimulate a second rise in cAMP, independent of LH. This study examined the temporal relationships among PGE2, PGF2 alpha 6-keto-PGF1 alpha, estradiol-17 beta, progesterone, testosterone, androstenedione and the biphasic increases of cAMP in follicles of rabbits. Does received indomethacin (IN, 20 mg/kg, i.v.; n = 30) or phosphate buffer (C; n = 30), 0.5 h before 50 ug of LH. At laparotomy at 0, 0.5, 1, 2, 4 or 8 h after LH, blood was collected from each ovarian vein and two follicles per ovary were aspirated of fluid and excised. Plasma and follicular tissue and fluid were assayed for PG and steroids. Tissue and fluid were assayed for cAMP. In C does, cAMP (pmol/follicle) in tissue increased from 11.3 at 0 h to 14.2 at 0.5 h, decreased at 1 h (5.4) and increased linearly through 8 h to 14.5. In IN-treated does, cAMP remained high from 0.5 (13.2) to 2 h (16.3), decreased at 4 h (7.9) then increased again by 8 h (15.5). Indomethacin decreased all PG in follicular tissue but 6-keto-PGF1 alpha rose after 2 h, whereas PGE2 and PGF2 alpha did not. Estradiol-17 beta, progesterone, and androstenedione did not vary with treatment; testosterone was increased (P less than .05) by IN. PGE2 or PGF2 alpha may terminate the first phase of cAMP production, rather than initiate the second phase.     

Regulation of prostaglandin biosynthesis by luteinizing hormone and bradykinin in rat preovulatory follicles in vitro.

Luteinizing hormone (LH) stimulates prostaglandin biosynthesis and steroidogenesis in preovulatory (PO) follicles prior to ovulation. Since the ovulatory process shares many similarities with an inflammatory reaction, mediators of the inflammatory response, such as bradykinin (BK) have been suggested to modulate the effects of LH. In the present study the effect of BK (5 microM) on: 1) prostaglandin biosynthesis (PGE2, PGF2 alpha and 6-keto-PGF1 alpha), 2) the levels of two enzymes in the cyclo-oxygenase pathway, prostaglandin endoperoxide synthase (PGS) and prostacyclin synthase (PCS), and 3) cyclic adenosine 3'5'-monophosphate (cAMP) and progesterone response of PO follicles incubated in vitro were examined. LH (0.1 microgram/ml) stimulated the accumulation of cAMP and progesterone in the medium, while BK had no effect on these parameters. BK exerted a slight stimulatory effect on PGE2, and PGF2 alpha, (p less than or equal to 0.01) but not on 6-keto-PGF1 alpha synthesis, but no changes in PGS or PCS levels could be detected. The effect of LH on prostaglandin biosynthesis was much more pronounced, with an increase of PGE2, PGF2 alpha and 6-keto-PGF1 alpha. LH also induced PGS. The combination of LH and BK did not alter these responses compared to that of LH alone. This study demonstrates that BK stimulates prostaglandin biosynthesis in PO follicles. In contrast to LH, this effect of BK does not seem to involve the adenylate cyclase system, since BK did not stimulate cAMP production. BK did not affect the levels of PGS or PCS, and the stimulatory effect of BK is suggested to involve an increase in the availability of substrate for the cyclo-oxygenase pathway.     

Effect of modulators of cytoskeletal function on desensitization and recovery of PGE2-responsive ovarian adenylate cyclase.

Exposure of cultured Graafian follicles to PGE2 for 20 h resulted in a loss of the cyclic AMP response to fresh hormone. This desensitization was prevented by addition to the medium of D2O (25--50%) or Li+ (0.6--6 mM), agents believed to stabilize microtubules, as well as by phalloidin (1.0--10 microM), believed to stabilize the polymerized state of actin, in a dose-dependent manner. The spontaneous recovery of responsiveness to PGE2 upon incubation of refractory follicles for 6 h in hormone-free medium was prevented by addition to the medium of cytochalasin B (CB; 3 microgram/ml) or of the actin-binding myosin subfragment HMM S-1 (80 microgram/ml) or of anti-actin serum; viz. by agents likely to interfere with microfilament function. D2O (50%) caused morphological damage to the inner layer of the membrana granulosa and severe depression of protein synthesis. The other drugs used (phalloidin, LiCl and cytochalasin B) had no such effects. Resensitization of refractory follicles was also prevented by cycloheximide (10 micrograms/ml) and by actinomycin D (10 micrograms/ml). It is speculated that the recovery process may involve the insertion of a newly synthesized protein, such as PG-receptor, into the membrane by a mechanism dependent on microfilament action. These findings provide suggestive evidence for the hypothesis that cytoskeletal elements associated with the cell membrane take part in the modulation of the adenylate cyclase response to hormones.     

Effect of gonadotropins and prostaglandin on cumulus mucification in cultures of intact follicles

Follicles of 28 day-old pregnant mare serum gonadotropin (PMSG)-primed rats, were cultured for up to 24 hours in the presence or absence of ovine gonadotropins, highly purified rat gonadotropins, dibutyryl cyclic AMP (dbcAMP), methylisobutylxanthine (MIX), choleratoxin (CT), or prostaglandin E2 (PGE2). The morphology of the cumulus-oocyte complexes isolated from these follicles was subsequently examined with the light microscope. Cumulus mucification was studied under the different culture conditions using scanning electron microscopy (SEM) and the hyaluronidase sensitivity test. The features of the cumulus-oocyte complexes in the control cultures did not change throughout the incubation period, while complexes from follicles incubated with LH, FSH, dbcAMP, MIX, CT, or PGE2 changed their appearance and accumulated extracellular mucoid material. Treatment of these cumuli with hyaluronidase resulted in lysis of the extracellular mucus and dispersal of the cumulus masses. The results of this study agree with our earlier observation that the maturation of the cumulus-oophorus, which occurs in vivo following the LH surge, can be induced in vitro by either gonadotropins or cAMP. Prostaglandin E2 did not affect cumulus cells, unless incubated enclosed by their follicles. This suggests that this hormone may influence the cumulus cells indirectly, probably via other components.     

Desensitization of testicular ornithine decarboxylase to prostaglandin E2

Intratesticular injection of prostaglandin E2 at a dose of 10 or 25 micrograms per testis caused desensitization of the testis to ornithine decarboxylase activity at 24 h after the injection. PGE2 caused desensitization in both Leydig cells and seminiferous tubules. The desensitized testis was refractory to follicle stimulating hormone, luteinizing hormone and cAMP in addition to PGE2. These results indicate that testicular desensitization to PGE2 is at a step beyond cAMP formation.     

Aspartic acid 564 in the third cytoplasmic loop of the luteinizing hormone/choriogonadotropin receptor is crucial for phosphorylation-independent interaction with arrestin2

Arrestin2 binding to the active but unphosphorylated luteinizing hormone/choriogonadotropin receptor (LH/CG R) in ovarian follicles is triggered by activation of ADP-ribosylation factor 6 (ARF6) and leads to uncoupling of this receptor from cAMP signaling. We sought to determine how arrestin2 binds to LH/CG R, if binding is of high affinity, and if the receptor also binds arrestin3. Desensitization of intact LH/CG R was equally sensitive to ectopic constructs of arrestin2 that bind other G protein-coupled receptors (GPCRs) either in a phosphorylation-independent or -dependent manner. Intact LH/CG R was not desensitized by ectopic arrestin3 constructs. Surface plasmon resonance studies showed that arrestin2 bound a synthetic third intracellular (3i) LH/CG R loop peptide with picomolar affinity; arrestin3 bound with millimolar affinity. To determine whether Asp-564 in the 3i loop mimicked the phosphorylated residue of other GPCRs, human embryonic kidney (HEK) cells were transfected with wild-type (WT) and D564G LH/CG R. An agonist-stimulated ARF6-dependent arrestin2 undocking pathway to drive desensitization of WT receptor was recapitulated in HEK cell membranes, and ectopic arrestin2 promoted desensitization of WT LH/CG R. However, D564G LH/CG R in HEK cells was not desensitized, and synthetic 3i D564G peptide did not bind arrestin2. Synthetic 3i loop peptides containing D564E, D564V, or D564N also did not bind arrestin2. We conclude that the ARF6-mediated mechanism to release a pool of membrane-delimited arrestin to bind GPCRs may be a widespread mechanism to deliver arrestin to GPCRs for receptor desensitization. Unlike other GPCRs that additionally require receptor phosphorylation, LH/CG R activation is sufficient to expose a conformation in which Asp-564 in the 3i loop confers high affinity binding selectively to arrestin2.     

Gonadotropic regulation of prostaglandin production by ovarian follicular cells of the pig

Prepubertal gilts were treated with 750 IU pregnant mare's serum gonadotropin (PMSG) and 72 h later with 500 IU human chorionic gonadotropin (hCG) to induce follicular growth and ovulation. Dispersed granulosa cells (GC) and theca interna cells (TC) from follicles of gilts 72 h (GC-72 and TC-72, respectively) and 108 h (GC-108 and TC-108 h, respectively) after PMSG treatment were cultured for 0, 12, 24, and 36 h in medium with or without luteinizing hormone (LH), dibutyryl cyclic adenosine 3',5'-monophosphate [Bu)2cAMP), calcium ionophore (A23187), and/or arachidonic acid (AA), and the production of prostaglandin E2 (PGE) and prostaglandin F2 alpha (PGF) was measured by radioimmunoassay. TC-72 was the principal source of PGs 72 h after PMSG. At 108 h, the production of PGE and PGF by GC was increased 10- and 30-fold, respectively, whereas corresponding increases by TC were 2-fold. LH and A23187 significantly stimulated PGE and PGF production by both GC-72 and TC-72, but only thecal PG production was stimulated by (Bu)2cAMP. LH had minimal or no effect on PG production by GC-108 and TC-108, but A23187 (GC-108, TC-108) and (Bu)2cAMP (TC-108) were stimulatory. Basal PG production by GC-72, GC-108, and TC-108 was stimulated by AA. However, production by GC and TC cultured in medium containing AA and LH, A23187, or (Bu)2cAMP was not different from that produced by AA alone. These findings suggested that GC and TC can synthesize PGs in vitro, but AA availability is rate-limiting in GC. After exposure to hCG in vivo, the capacity of both cell types to produce PGs is increased but is limited by AA availability.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Effect of alterations in pulsatile luteinizing hormone release on ovarian follicular atresia and steroid secretion on diestrus 1 in the rat estrous cycle

This study examined the importance of pulsatile luteinizing hormone (LH) release on diestrus 1 (D1; metestrus) in the rat estrous cycle to ovarian follicular development and estradiol (E2) secretion. Single injections of a luteinizing hormone-releasing hormone (LHRH) antagonist given at -7.5 h prior to the onset of a 3-h blood sampling period on D1 reduced mean blood LH levels by decreasing LH pulse amplitude, while frequency was not altered. Sequential injections at -7.5 and -3.5 h completely eliminated pulsatile LH secretion. Neither treatment altered the total number of follicles/ovary greater than 150 mu in diameter, the number of follicles in any size group between 150 and 551 mu, or plasma E2, progesterone, or follicle-stimulating hormone (FSH) levels. However, both treatments with LHRH antagonist significantly increased the percentage of atretic follicles in the ovary. These data indicate that: 1) pulsatile LH release is an important factor in determining the rate at which follicles undergo atresia on D1; 2) reductions in LH pulse amplitude alone are sufficient to increase the rate of follicular atresia on D1; 3) an absence of pulsatile LH release for a period of up to 10 h on D1 is not sufficient to produce a decline in ovarian E2 secretion, most likely because the atretic process was in its early stages and had not yet affected a sufficient number of E2-secreting granulosa cells to reduce the follicle's capacity to secrete E2; and 4) suppression or elimination of pulsatile LH release on D1 is not associated with diminished FSH secretion.     

Dose-related effects of luteinizing hormone on the pattern of steroidogenesis and cyclic adenosine monophosphate release in superfused preovulatory rat follicles

The secretion of steroids and the release of cAMP in response to repeated luteinizing hormone (LH) stimulation were examined during superfusion of isolated preovulatory rat follicles. A high dose of ovine LH (1 microgram/ml for 20 min) caused a prolonged increase in the secretion of progesterone (P) and 20 alpha-dihydroprogesterone (20 alpha-OHP) and a transient increase in the secretion of testosterone (T) and estradiol-17 beta (E2), and was accompanied by a peak of cAMP release. A single pulse of LH at a low dose level (10 mg/ml for 20 min) gave a limited increase in T secretion, but no clear change in P, 20 alpha-OHP and E2 secretion or cAMP release. When the follicles were challenged with a second pulse of LH (at 1 microgram/ml), the response varied according to the dose of LH delivered in the preceding pulse. Following exposure to the high dose of LH, the follicles were partially refractory to the second LH challenge in terms of cAMP and P and the secretion of T and E2 remained low. The low dose of LH, however, had a conditioning effect on the follicles since the response to the second LH challenge was amplified in terms of P, 20 alpha-OHP and cAMP. In this case a secondary increase in T and E2 secretion was found. The differential response to varying doses of LH are likely to reflect the physiological control of steroidogenesis during final follicular maturation.     

Involvement of prostaglandins in LH-induced superovulation in the cyclic hamster

Osmotic minipumps containing 400 micrograms ovine LH were inserted subcutaneously (sc) on day 1 (estrus) at 09:00-10:00h of the cycle in the hamster. This treatment induced increased ovarian blood flow by day 3 and superovulation of 30.0 +/- 1.4 ova at the next estrus compared to controls (16.5 +/- 0.8 ova). The continuous infusion of LH throughout the cycle increased prostaglandin F (PGF) and decreased prostaglandin E (PGE) in the growing follicles destined to ovulate and suppressed a day 3 increase in PGF concentrations in the nonluteal ovarian remnant devoid of the larger follicles. Indomethacin, a cyclooxygenase inhibitor, given sc (2 or 4 mg regimens) at 12:00-14:00h on days 1 and 2, at 09:00h and 17:00h on day 3 and at 09:00h on day 4 of the cycle to LH-infused and saline treated animals suppressed ovarian prostaglandin levels, prevented the superovulation and prevented the increased ovarian blood flow. Exogenous PGF2 alpha or PGE2 restored the superovulatory effect of LH infusion in the presence of indomethacin. The results suggest that the superovulation in response to continuous LH infusion may be mediated in part by prostaglandins via altered ovarian blood flow.     

Induction of maturation in follicle-enclosed oocytes: the response to gonadotropins at different stages of follicular development

Antral follicles, isolated from either nontreated or pregnant mare's serum gonadotropin (PMSG)-primed 27-day-old rats, were incubated in the absence or the presence of either luteinizing hormone (LH), follicle-stimulating hormone (FSH), or forskolin. The effect of these agents on oocyte maturation and cyclic adenosine 3',5'-monophosphate (cAMP) accumulation was studied and compared. Both gonadotropins, LH and FSH, as well as forskolin, effectively induced maturation of oocytes enclosed by large antral follicles isolated from PMSG-primed rats. On the other hand, we found that maturation of oocytes enclosed by small antral follicles, isolated from nonprimed and PMSG-primed rats, could be induced by either FSH or forskolin but not by LH. cAMP determinations revealed that, in spite of the inability of LH to induce oocyte maturation, elevated concentrations of the nucleotide were detectable in small antral follicles exposed to this gonadotropin. Since granulosa cells isolated from the large but not the small antral follicles were stimulated by LH to generate cAMP, the elevation of cAMP concentrations in the small antral follicle apparently represented the response of the theca cells to this gonadotropin. Since it is the ability of the granulosa cells to interact with the hormone that determines whether or not oocyte maturation will occur, we suggest that the granulosa, but not the theca cells, mediate LH action to induce oocyte maturation.     

The role of prostaglandins in the development of refractoriness to LH stimulation by Graafian follicles.

The possibility that prostaglandins might be responsible for the development of the pre-ovulatory refractoriness to the stimulation by LH of cyclic AMP accumulation in vitro in the Graafian follicle was examined. Isolated rabbit Graafian follicles were obtained at estrus and at 0.5, 5 and 9 hours after an ovulatory dose of LH. The follicles were incubated in vitro in the presence of (8-3H)adenine and the accumulation of (8-3H)cyclic AMP measured. Follicles from estrous animals responded to the in vitro addition of LH with a marked increase of cyclic AMP accumulation and lost this response as the time of ovulation approached. Animals pretreated with indomethacin, which inhibits the usual pre-ovulatory rise of follicular prostaglandin levels, showed essentially the same loss of responsiveness. Indomethacin alone was without effect. It is concluded that prostaglandins are not the major factor in the development of refractoriness to LH stimulation in vitro which has been observed in pre-ovulatory follicles.     

Steroid-independent effect of gonadotropins on prostaglandin synthesis in rat Graafian follicles in vitro.

The content of prostaglandins of the E-group (PGE) or F-group (PGF) was determined by radioimmunoassay in rat ovaries and in homogenates of cultured Graafian follicles. Intraperitoneal administration of luteinizing hormone (NIH-LH-S18; 10 mug/rat) at 9.00 h on any day of the estrous cycle caused an increase in ovarian PGE content within 5 h. The response was greatest on the day of proestrus (940% rise), i.e. when the ovary contains large follicles, and least at metestrus (80%). Follicles explanted from proestrous rats before the preovulatory gonadotropin surge responded to addition of LH (1-5 mug/ml) to the culture medium with a 10 to 30-fold increase in PGE and a 5-fold increase in PGF accumulation over a 5-h-period. Follicle stimulating hormone (NIH-FSH-S9; 10 mug/ml) caused a similar rise in follicular PGE accumulation, even after treatment of the FSH preparation with excess of an antiserum to the beta-subunit of LH. Stimulation of follicular PG accumulation was unimpaired during suppression of progesterone and estrogen synthesis by aminoglutethimide. It is concluded that these steroids play no part in the mediation of the LH-effect on follicular prostaglandin formation.     

Inhibition of ovulation in rabbits by intrafollicular injection of indomethacin and prostaglandin F antiserum

Indomethacin, an inhibitor of prostaglandin biosynthesis, was 100% effective in blocking luteinizing hormone (LH)-induced ovulation when administered via microinjection directly into 22 follicles in twelve rabbits, 5 hours after intravenous injection of the gonadotropin. Ovulation was similarly blocked in 24 of 25 follicles injected with antiserum prepared against prostaglandin F_2α. Antiserum against prostaglandin E₂, at the same dosage (100 μg lyophilized serum per follicle), was considerably less effective, preventing ovulation in only 6 of 14 follicles. Control follicles injected with the phosphate buffer vehicle, or with normal rabbit serum, underwent normal ovulation and luteinization. LH injection caused a striking increase in concentration of F-type prostaglandins in follicles shortly before ovulation, an increase which was prevented by i.v. or intrafollicular injection of ovulation-blocking blocking dosages of indomethacin. These findings provide evidence in support of a role for prostaglandins, acting at the follicular level, in the process of ovulation.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE2 at a dose of 10 microgram per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE2 and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE2 in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 microgram/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE2 and PGF2 alpha stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.