Post ovulatory changes in the concentration of prostaglandins in rabbit Graafian follicles

In previous studies we have demonstrated that prior to hCG induced ovulation the levels of PGF and PGE in rabbit Graafian follicles increase markedly as ovulation approaches. We have now extended the study to include follicles obtained from animals at ovulation time and up to 48 hours after hCG injection. We have found that PGF reaches a maximum in ovulated follicles at the time of ovulation and then quickly decreases, whereas PGE continues to rise for several hours and then declines. The increase in both prostaglandins is limited to the follicles that actually ovulate. These data further document the proposed role for prostaglandins in the ovulatory process.     

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.     

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 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 the presence of [8-³H]adenine and the accumulation of [8-³H]cyclic AMP measured. Follicles from estrous animals responded to the 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 which has been observed in pre-ovulatory follicles.     

Prostaglandin F production by granulosa cells from rabbit pre-ovulatory follicles

The concentration of PGF in rabbit Graafian follicles increases at ovulation but the cell type responsible for PGF secretion has not been identified. We have found that a pure population of granulosa cells isolated from pre-ovulatory follicles of estrous rabbits secrete prostaglandin F in tissue culture (total secretion, 446 ng/10 days; 0.09 pg/cell/day). LH/FSH did not influence the rate of PGF secretion, but there was a 50% inhibition after dibutyryl cAMP treatment, and complete inhibition by indomethacin. These results indicate that granulosa cells could secrete the prostaglandin which accumulates in the follicle at ovulation, and that PGF secretion may be modified by the addition of cAMP to the medium.     

Prostaglandin F production in vitro by granulosa cells from rabbit pre-ovulatory follicles

The concentration of PGF in rabbit Graafian follicles increases at ovulation but the cell type responsible for PGF secretion has not been identified. We have found that a pure population of granulosa cells isolated from pre-ovulatory follicles of estrous rabbits secrete prostaglandin F in tissue culture (total secretion, 446 ng/10 days; 0.09 pg/cell/day). LH/FSH did not influence the rate of PGF secretion, but there was a 50% inhibition after dibutyryl cAMP treatment, and complete inhibition by indomethacin. These results indicate that granulosa cells could secrete the prostaglandin which accumulates in the follicle at ovulation, and that PGF secretion may be modified by the addition of cAMP to the medium.     

Pre and post ovulatory changes in the concentration of prostaglandins in rat graafian follicles.

The concentrations of prostaglandin F (PGF) and prostaglandin E (PGE) were measured by radioimmunoassay in isolated Graafian follicles of mature female rats during the pre and post ovulatory period of the estrous cycle. The levels of these prostaglandins were low and relatively constant from 8 a.m. to 4 p.m. on the day of proestrus, but there was a marked increase at 8 p.m. of proestrus reaching an apparent maximum at midnight (PGF 18 fold, PGE 70 fold). By 4 a.m. to 8 a.m. on the morning of estrus these prostaglandins declined rapidly to levels similar to those observed between 8 a.m. and 4 p.m. on the day of proestrus. The increases in prostaglandin levels occurred after the LH peak and apparently before the time of ovulation. These data confirm the role of PGF and PGE in the local mechanism of ovulation in the normal adult of a spontaneously ovulating animal species.     

Localization and in vitro synthesis of prostaglandins in components of rabbit preovulatory graafian follicles.

Graafian follicles obtained 9 hours after the injection of human chorionic gonadotropin (hCG) into mature rabbits were dissected into a follicular fluid component, a granulosa cell-oocyte component, and a residual wall component, (the latter containing mostly theca tissue with a small and variable amount of adhering granulosa cells). The amounts of PGE and PGF were determined for each component. The follicular fluid contained approximately 4-10 times more PGE and PGF than either the granulosa cell-oocyte component or the residual wall component. The latter two components contained approximately equal amounts of these prostaglandins. The in vitro biosynthesis of PGE and PGF was also studied and it was found that the granulosa cell-oocyte component had about 4 fold the capacity of the residual wall, and that the follicular fluid synthesized no prostaglandins. There was no significant effect of LH on either PGE or PGF synthesis in any of the components.     

Interactive roles of progesterone, prostaglandins, and collagenase in the ovulatory mechanism of the ewe

Interrelationships between production of progesterone (P4), prostaglandin (PG) E2 and PGF2 alpha, and collagenase by periovulatory ovine follicles and their possible involvements in the ovulatory process were investigated. Follicles were isolated from ovaries at intervals (0 to 24 h) after the initiation of the preovulatory surge of luteinizing hormone (LH). Progesterone and PGs within follicles were determined by radioimmunoassay. Digestion of radioactive collagen during coincubation with tissue homogenates was used to assess the production of a bioactive follicular collagenase(s). Follicular accumulation of PGs and P4 increased at 12 and 16 h, respectively, after the onset of the surge of LH; PGE2 then decreased at 20 h. Collagenolytic activity of follicular tissue increased at 20 h and was maximal at 24 h (during the time of follicular rupture). An inhibitor of synthesis of P4 (isoxazol) or PGs (indomethacin) was injected into the follicular antrum at 8 h. Isoxazol did not prevent the initial rise in PGs, but inhibited synthesis of PGF2 alpha at 16 h and therafter. Isoxazol negated the decline in PGE2 and increase in collagenolysis. Indomethacin did not influence synthesis of P4; however, it suppressed collagenolytic activity of follicular tissue. Ovaries with treated follicles were left in situ and observed for an ovulation point at 30 h. Isoxazol or indomethacin was a potent inhibitor of ovulation. The blockade of ovulation by isoxazol was reversed by systemic administration of P4 or PGF2 alpha, but not by PGE2. Reversal of the blockade by indomethacin was accomplished with PGE2 or PGF2 alpha. Collagenolytic activity of follicular tissue was likewise restored by such treatments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pre-ovulatory changes in cyclic AMP and prostaglandin concentrations in follicular fluid of gilts.

The concentrations of cyclic adenosine 3', 5'-monophosphate (cyclic AMP) and prostaglandins E and F (PGE and PGF) were determined in follicular fluid collected from follicles of prepubertal gilts at various times after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induced ovulation. The concentrations of cyclic AMP, PGE and PGF in the follicular fluid after PMSG treatment but prior to hCG injection were about 1 pmol/ml, 1 ng/ml and 0.2 ng/ml, respectively. After hCG administration, the follicular fluid levels of cyclic AMP increased markedly, reaching a peak (400-fold increase) about 4 h after injection and then declined gradually to pre-hCG levels. A second rise (2.5- to 5-fold increase) occurred about 30 h after hCG with the levels being sustained up to the expected time of ovulation. In contrast, the levels of PGE and PGF remained relatively constant until 28-30 h after hCG treatment. Thereafter, the concentrations of both prostaglandins began to rise with the increases becoming more pronounced and reaching maximal values as the expected time of ovulation approached. These data provide further evidence for a physiological role of follicular prostaglandins in the process of ovulation but do not support an obligatory role for prostaglandins in the acute gonadotropin stimulation of cyclic AMP formation.     

Pre-ovulatory changes in cyclic AMP and prostaglandin concentrations in follicular fluid of gilts

The concentrations of cyclic adenosine 3′,5′-monophosphate (cyclic AMP) and prostaglandins E and F (PGE and PGF) were determined in follicular fluid collected from follicles of prepubertal gilts at various times after treatment with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) to induce ovulation. The concentrations of cyclic AMP, PGE and PGF in the follicular fluid after PMSG treatment but prior to hCG injection were about 1 pmol/ml, 1 ng/ml and 0.2 ng/ml, respectively. After hCG administration, the follicular fluid levels of cyclic AMP increased markedly, reaching a peak (400-fold increase) about 4 h after injection and then declined gradually to pre-hCG levels. A second rise (2.5- to 5-fold increase) occurred about 30 h after hCG with the levels being sustained up to the expected time of ovulation. In contrast, the levels of PGE and PGF remained relatively constant until 28–30 h after hCG treatment. Thereafter, the concentrations of both prostaglandins began to rise with the increases becoming more pronounced and reaching maximal values as the expected time of ovulation approached. These data provide further evidence for a physiological role of follicular prostaglandins in the process of ovulation but do not support an obligatory role for prostaglandins in the acute gonadotropin stimulation of cyclic AMP formation.     

Comparison of the effect of nonsteroidal and steroidal antiinflammatory agents on prostaglandin production during ovulation in the rabbit

The antiinflammatory agents diclofenac, fenoprofen and aspirin were tested to determine how well they inhibit the pre-ovulatory elevation in prostaglandin (PG) production in rabbit follicles in comparison to indomethacin. In addition, the antiinflammatory agent dexamethasone and the antipyretic agent acetaminophen were tested. The agents were administered 8 h after the ovulatory process was stimulated by hCG (50 I.U./kg). At 10 h after hCG (i.e., at the expected time of ovulation) control follicles had PGF and PGE levels of 370.0 and 582.6 pg/mg of follicle, respectively. Diclofenac inhibited PG production the most-reducing PGF and PGE to 22.8 and 53.6 pg/mg, respectively. Indomethacin reduced the PGF and PGE levels to 27.4 and 76.6 pg/mg, respectively. Fenoprofen was less effective, reducing the PGF and PGE to 77.8 and 222.4 pg/mg, respectively. Aspirin reduced the PGF and PGE to 123.4 and 174.6 pg/mg, respectively. Dexamethasone and acetaminophen did not inhibit PG production. Ovulation was completely inhibited by diclofenac and indomethacin, partially inhibited by fenoprofen, and unaffected by aspirin, acetaminophen, or dexamethasone. The results suggest that any potent antiinflammatory agent can inhibit ovulation provided it adequately reduces PG production; whereas antiinflammatory agents are ineffective. The anti-inflammatory agent must completely abolish the preovulatory elevation in PGs in mature follicles in order to totally inhibit ovultion.     

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.     

Steroid production by the isolated rabbit ovarian follicle.

Graafian follicles from New Zealand white rabbits were incubated at 37 degrees C for various periods of time with air as the gas phase. Media were changed every 15 min and stored at -15 degrees C until analysed for progestins, 17 beta-hydroxyandrogens and oestrogens using established radioimmunoassay procedures. At fixed times after the start of the incubations, media containing various test substances were added with subsequent replacement by medium alone. Addition of 5 mug LH/ml for 1 sec caused a dramatic increase in the synthesis and secretion of androgen with lesser increases in progestin and oestrogen. Puromycin and cycloheximide but not actinomycin D, inhibited LH-induced steroidogenesis. Cycli AMP, dibutyryl cycli AMP, cyclic CMP, 5'-AMP, and theophylline also caused an increase in androgen production which rapidly ceased when media without nucleotides were added. Sodium fluoride had no effect on steroidogenesis. From these data it was concluded that (i) the rabbit follicle is the major source of ovarian androgen; (ii) the binding of LH to the follicular cells is a rapid process; (iii) the events following LH binding do not require the presence of LH in the medium; (iv) cyclic nucleotides which may act as second messengers also stimulate steroidogenesis; (v) the effects of LH and cyclic nucleotides on steroidogenesis are different; and (vi) the action of LH on follicular steroidogenesis probably occurs in the translational level.     

Stimulation of prostaglandin E production by concanavalin A in isolated graafian follicles

Addition of Concanavalin A (con A) to isolated rat Graafian follicles induced prostaglandin E (PGE) production after 2 h of incubation. PGE synthesis continued throughout 24 h culture period. Cyclic AMP accumulation was noted after 6 h of incubation with Con A. Aspirin, indomethacin and flufenamate prevented both the stimulation of PGE production and of cyclic AMP accumulation by Con A; antibodies to PGE prevented the cyclic AMP production. These studies indicate that the interaction of Con A with the follicle results in PGE production. It seems that besides the known pathway for the induction of PGE synthesis in the ovarian follicle, via elevation of intracellular cyclic AMP, an additional pathway, via an external signal which is independent of cyclic AMP exists.     

Localization and in vitro synthesis of prostaglandins in components of rabbit preovulatory Graafian follicles

Graafian follicles obtained 9 hours after the injection of human chorionic gonadotropin (hCG) into mature rabbits were dissected into a follicular fluid component, a granulosa cell-oocyte component, and a residual wall component, (the latter containing mostly theca tissue with a small and variable amount of adhering granulosa cells). The amounts of PGE and PGF were determined for each component. The follicular fluid contained approximately 4–10 times more PGE and PGF than either the granulosa cell-oocyte component or the residual wall component. The latter two components contained approximately equal amounts of these prostaglandins. The $\text{in vitro}$ biosynthesis of PGE and PGF was also studied and it was found that the granulosa cell-oocyte component had about 4 fold the capacity of the residual wall, and that the follicular fluid synthesized no prostaglandins. There was no significant effect of LH on either PGE or PGF synthesis in any of the components.     

Pre-ovulatory changes in follicular fluid prostaglandin F levels in swine

The concentration of prostaglandin F (PGF) has been measured by radioimmunoassay in follicular fluid collected from follicles at various time intervals after treatment of prepuberal gilts with pregnant mare serum gonadotropin and human chorionic gonadotropin to induce ovulation. A high proportion of animals will ovulate 116 ± 8 hr after this treatment. Pre-ovulatory follicles can be identified on the basis of gross morphological appearance 10–12 hr before the predicted time of ovulation. The concentration of PGF in fluid from follicles judged not to be pre-ovulatory was relatively constant at about 0.45 ng per g and appeared to be independent of the time of sampling. An increase in the concentration of PGF was observed in fluid collected from follicles classified as destined to ovulate. This increase became more pronounced as the time of ovulation approached and reached a maximum at or about the time of follicle rupture.These data provide evidence in support of a role for prostaglandins in the ovulatory process in the pig.     

Stimulation of prostaglandin E production by concanavalin A in isolated Graafian follicles

Addition of Concanavalin A (Con A) to isolated rat Graafian follicles induced prostaglandin E (PGE) production after 2 h of incubation. PGE synthesis continued throughout 24 h culture period. Cyclic AMP accumulation was noted after 6 h of incubation with Con A. .Aspirin, indomethacin and flufenamate prevented both the stimulation of PGE production and of cyclic AMP accumulation by Con A; antibodies to PGE prevented the cyclic AMP production. These studies indicate that the interaction of Con A with the follicle results in PGE production. It seems that besides the known pathway for the induction of PGE synthesis in the ovarian follicle, via elevation of intracellular cyclic AMP, an additional pathway, via an external signal which is independent of cyclic AMP exists.     

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.     

Pre and post ovulatory changes in the concentration of prostaglandins in rat Graafian follicles

The concentrations of prostaglandin F (PGF) and prostaglandin E (PGE) were measured by radioimmunoassay in isolated GRaafian follicles of mature female rats during the pre and post ovulatory period of the estrous cycle. The levels of these prostaglandins were low and relatively constant from 8 a.m. to 4 p.m. on the day of proestrus, but there was a marked increase at 8 p.m. of proestrus reaching an apparent maximum at midnight (PGF 18 fold, PGE 70 fold). By 4 a.m. to 8 a.m. on the morning of estrus these prostaglandins declined rapidly to levels similar to those observed between 8 a.m. and 4 p.m. on the day of proestrus. The increases in prostaglandin levels occurred after the LH peak and apparently before the time of ovulation. These data confirm the role of PGF and PGE in the local mechanism of ovulation in the normal adult of a spontaneously ovulating animal species.     

Modulators of Bufo arenarum ovulation

In this study we investigated ovulation in vitro using ovary samples from Bufo arenarum with respect to their response to stimulation with homologous pituitary homogenate (HPH) or with progesterone and prostaglandins (PGF2alpha and PGE1) as intermediates of pituitary action. Ovary samples were obtained from animals captured during the breeding period. Our results demonstrate that the ovulatory response to all different inducers was dose dependent, the highest percentage of ovulated oocytes being obtained with HPH treatment. An important increase in the ovulatory response was obtained by the association of PGF2alpha with either HPH or progesterone at suboptimal doses, indicating that this prostaglandin induced a synergistic potentiating effect. Incubation with cyclooxygenase inhibitors (indomethacin or diclofenac sodium) produced a significant decrease in the ovulation induced by HPH, demonstrating that prostaglandins are involved in the action of the pituitary gland in this process. According to our results, PGE1 not only had no participation in the ovulatory process, but also produced an inhibitory effect on ovulation induced by HPH treatment.