The role of protein synthesis in the stimulation by LH of prostaglandin accumulation in rat preovulatory follicles in vitro

Preovulatory follicles isolated from immature rats, treated $\text{in vivo}$ with pregnant mare's serum gonadotropin, were incubated $\text{in vitro}$ and the accumulation of prostaglandin E measured. The addition of luteinizing hormone (5 μg/ml) increased this accumulation, after a lag period of 3 hours. This delay suggested the involvement of macromolecular synthesis in the mechanism of prostaglandin stimulation by luteinizing hormone. When the synthesis of protein was inhibited by the addition of puromycin (100 μM), the luteinizing hormone stimulation of prostaglandin E in these follicles was completely abolished. This inhibition was not seen with an analogue of puromycin, which does not inhibit protein synthesis, puromycin amino-nucleoside. These data suggest that concomitant protein synthesis is required for the luteinizing hormone stimulation of prostaglandin accumulation in rat follicles.     

Multifactorial regulation of prostaglandin synthesis in preovulatory goldfish ovarian follicles.

Goldfish preovulatory ovarian follicles (prior to germinal vesicle breakdown) were utilized for studies investigating the actions of activators of different signal transduction pathways on prostaglandin (PG) production. The protein kinase C (PKC) activators phorbol 12-myristate 13-acetate (PMA; 100-400 nM), 1-oleoyl-2-acetylglycerol (5 and 25 micrograms/ml), and 1,2-dioctanoylglycerol (10 and 50 micrograms/ml) stimulated PGE production; the inactive phorbol 4 alpha-phorbol didecanoate, which does not activate PKC, had no effect. Calcium ionophore A23187 (0.25-4.0 microM) stimulated PGE production and acted in a synergistic manner with activators of PKC. Although produced in lower amounts than PGE, PGF was stimulated by PMA and A23187. The direct activator of phospholipase A2, melittin (0.1-1.0 microM), stimulated a dose-related increase in PGE production, whereas chloroquine (100 microM), a putative inhibitor of phospholipase A2, blocked basal and PMA + A23187-stimulated PGE production. Several drugs known to elevate intracellular levels of cAMP including the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (0.1-1.0 mM), forskolin (10 microM), and dibutyryl cAMP (dbcAMP; 5 mM) attenuate PMA + A23187-stimulated PGE production. Melittin-stimulated production of PGE was inhibited by dbcAMP, suggesting that the action of cAMP was distal to the activation of phospholipase A2. In summary, these studies demonstrate that activation of PKC and elevation of intracellular calcium levels stimulate PG production, in part, through activation of phospholipase A2. The adenylate cyclase/cAMP signalling pathway is inhibitory to PG production by goldfish ovarian 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.     

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.     

Dexamethasone inhibits luteinizing hormone-induced synthesis of steroidogenic acute regulatory protein in cultured rat preovulatory follicles

The effect of dexamethasone on LH-induced synthesis of steroidogenic acute regulatory (StAR) protein was studied in a serum-free culture of preovulatory follicles. StAR protein is a steroidogenic tissue-specific, hormone-induced, rapidly synthesized protein previously shown to be involved in the acute regulation of steroidogenesis, probably by promoting the transfer of cholesterol to the inner mitochondrial membrane and the cytochrome P450 side-chain cleavage (P450(scc)) enzyme. Treatment of preovulatory follicles dissected from ovaries of cyclic adult rats on the morning of proestrus with LH for 24 h resulted in a dose-dependent increase in the level of StAR protein that reached a maximum at 10 ng LH/ml. This increase was associated with an increase in progesterone production. Treatment of the follicles with increasing concentrations (1-1000 ng/ml) of dexamethasone suppressed LH (10 ng/ml)-induced StAR protein levels and progesterone production in a dose-dependent manner. The amount of P450(scc) was not affected by this dexamethasone treatment, indicating that the loss of steroidogenic capacity was not a result of inhibition of P450(scc). Dexamethasone also decreased StAR protein levels and progesterone production induced by the adenylate cyclase activator forskolin (10(-5) M) or a cAMP analogue 8-Br-cAMP (0.5 mM). The effects of dexamethasone on 8-Br-cAMP-induced StAR protein levels and progesterone production were blocked by cotreatment of the follicles with glucocorticoid receptor antagonist RU-486. These results demonstrate that dexamethasone inhibits the LH-induced StAR protein levels and that the effects of dexamethasone are mediated by the glucocorticoid receptor.     

Effect of LH on progesterone and oestradiol production in vivo and in vitro by preovulatory rat follicles

Temporal changes in follicular oestradiol production induced in vitro and in vivo by LH were studied. In-vitro changes were measured by incubating preovulatory rat follicles for 12 h, changing the medium every 2 h. Follicles isolated at various intervals after an injection of 10 i.u. hCG were incubated for 2 h to measure changes in oestradiol production in vivo. In both studies there was an increase in oestradiol production lasting 4 h followed by a sharp decline. Progesterone production was also increased by LH in vitro or hCG in vivo, but remained high. A second exposure to LH did not raise oestradiol synthesis, but increased progesterone synthesis in vitro only. The decline in oestradiol production is most probably due to a decrease in C17-20 lyase activity, because addition of testosterone, but not of 17 alpha-hydroxyprogesterone, increased oestradiol production. Incubation of preovulatory follicles in the absence of LH or incubation of follicles derived from animals in which the spontaneous LH surge was blocked by an injection of pentobarbitone sodium also resulted in a decrease of oestradiol and an increase in progesterone production. This oestrogen-progesterone shift was also caused by a decrease in C17-20 lyase activity. The results demonstrate that the changes in steroid production in vivo and in vitro are similar and occur in the presence and absence of LH. It is concluded that the decrease in oestradiol production is dependent on the decrease in the activity of enzymes converting progesterone to aromatizable androgens.     

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.     

Changes in oestrogen-synthesizing ability of preovulatory bovine follicles relative to the peak of LH

Preovulatory bovine follicles (n = 28) were collected at different times after the onset of standing oestrus until shortly before ovulation. In-vitro conversion of tritiated androstenedione in the presence of NADPH by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) conversion into oestradiol-17 beta was high and production of oestrone was about 8-fold lower. During phases 1 (0-6 h after the LH peak) and 2A (6-14 h after the LH peak) the production of oestradiol and oestrone remained constant; the percentage of remaining androstenedione increased. In phase 2B (14-20 h after the LH peak) conversion into oestradiol and oestrone had decreased to about one third correlating with a higher percentage of remaining androstenedione. In phase 3 (20 h after the LH peak until ovulation) conversion into oestradiol and oestrone remained constant. The ratio between the production of oestrone and oestradiol remained constant throughout the phases of preovulatory development (0.13), indicating a concurrent inhibition of aromatase and 17 beta-hydroxysteroid dehydrogenase activities. Conversion into 19-hydroxyandrostenedione showed a pattern similar to that of oestradiol, and testosterone was produced in minute quantities. The results indicate that in preovulatory bovine follicles eventual inhibition of aromatization takes place at about 14 h after the preovulatory LH peak.     

Progesterone-synthesizing ability of preovulatory follicles of cows relative to the peak of LH

Preovulatory cow follicles (n = 34) were collected at different times after the onset of oestrus until shortly before ovulation. In-vitro conversion of tritiated pregnenolone in the presence of NAD+ by homogenates of the follicular wall was compared in phases relative to the LH peak. During phase 0 (before the LH surge) a moderate conversion into progesterone occurred, but it was subsidiary to that into 17 alpha-hydroxypregnenolone and other unidentified steroids. During phases 1 (0-6 h after the LH peak), 2A (6-14 h) and 2B (14-20 h) the production of progesterone and 17 alpha-hydroxypregnenolone remained constant; at phase 2B the percentage of remaining pregnenolone was higher than in the preceding phases. In phase 3 (20 h after the LH peak until ovulation) conversion into progesterone had increased about 4-fold to the highest levels observed (97% after 2 h incubation), and production of 17 alpha-hydroxypregnenolone and unidentified steroids was low. In an additional experiment, homogenates of the wall of 3 follicles at phase 3 were also incubated with tritated progesterone in the presence of NADPH. The percentage of remaining progesterone was high, and a moderate conversion into 17 alpha-hydroxyprogesterone occurred. In the main experiments, however, production of this steroid was not observed. The results indicate that steroid synthesis in the preovulatory follicle of the cow changes to the production of progesterone shortly before ovulation.     

Relationship between the preovulatory luteinizing hormone (LH) surge and androstenedione synthesis of preantral follicles in the cyclic hamster: detection by in vitro responses to LH

Preantral follicles of cyclic hamsters were isolated on proestrus, estrus and diestrus I, incubated for 3 h in 1 ml TC-199 containing 1 microgram ovine luteinizing hormone (LH) (NIH-S22), and the concentrations of progesterone (P), androstenedione (A) and estradiol (E2) determined by radioimmunoassay. At 0900-1000 h on proestrus (pre-LH surge) preantral follicles produced 2.4 +/- 0.3 ng A/follicle per 3 h, less than 100 pg E2/follicle and less than 250 pg P/follicle. At the peak of the LH surge (1500-1600 h) preantral follicles produced 1.8 +/- 0.2 ng P and 1.9 +/- 0.1 A and less than 100 pg E2/follicle. After the LH surge (1900-2000 h proestrus and 0900-1000 h estrus) preantral follicles were unable to produce A and E2 but produced 4.0 +/- 1.0 and 5.0 +/- 1.1 ng P/follicle, respectively. By 1500-1600 h estrus, the follicles produced 8.1 +/- 3.1 ng P/follicle but synthesized A (1.6 +/- 0.2 ng/follicle) and E2 (362 +/- 98 pg/follicle). On diestrus 1 (0900-1000 h), the large preantral-early antral follicles produced 1.9 +/- 0.3 ng A, 2.4 +/- 0.4 ng E2 and 0.7 +/- 0.2 ng P/follicle. Thus, there was a shift in steroidogenesis by preantral follicles from A to P coincident with the LH surge; then, a shift from P to A to E2 after the LH surge. The LH/follicle-stimulating hormone (FSH) surges were blocked by administration of 6.5 mg phenobarbital (PB)/100 g BW at 1300 h proestrus. On Day 1 of delay (0900-1000 h) these follicles produced large quantities of A (2.2 +/- 0.2 ng/follicle) and small amounts of E2 (273 +/- 27 pg/follicle) but not P (less than 250 pg/follicle).(ABSTRACT TRUNCATED AT 250 WORDS)     

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

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 μg/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 μg/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 μg/ml) caused a similar rise in follicular PGE accumulation, even after treatment of the FSH preparation with excess of an antiserum to the β-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.     

The possible involvement of prostaglandin F2 alpha in the stimulation of muscle protein synthesis by insulin

The addition of insulin (8 ng/ml) in vitro to muscles from fasted rabbits increased protein synthesis (+80%) to a value similar to that found in muscles from fed donors. The addition of either indomethacin or meclofenamate completely blocked this effect of insulin. Muscles from fasted rabbits released less prostaglandin (PG)F2 alpha into the medium and the presence of insulin increased and indomethacin and meclofenamate reduced PGF2 alpha release. Other conditions (work load and leucocyte pyrogen) which increase protein synthesis in muscle also stimulate PGF2 alpha release. As both arachidonic acid and PGF2 alpha in themselves increase protein synthesis we suggest that accelerated phospholipolysis and PG synthesis have a general role in the control of muscle protein turnover.     

Effects of ovarian hyperstimulation and isolated preovulatory follicles on LH responses to GnRH in rats

Adult rats were pretreated with a 3-day regimen of human menopausal gonadotrophin (hMG), PMSG, human FSH or hCG and experiments were carried out on the day of pro-oestrus. Treatment with hMG and hFSH induced a significant increase in the number of preovulatory follicles on the day of pro-oestrus and this was correlated with increased circulating concentrations of oestradiol. There was a parallel increase in the self-priming effect of GnRH, as observed from the biphasic LH response to a continuous GnRH challenge. PMSG treatment did not stimulate increased numbers of maturing follicles and was less effective in raising circulating oestrogen concentrations compared with hMG and hFSH. However, pituitary responsiveness was much higher after PMSG treatment and the biphasic response to continuous perfusion with GnRH was absent; LH release was high from the initiation of the stimulus. hCG alone failed to stimulate follicular maturation but enhanced pituitary LH responses. Hemi-pituitary glands perfused in the presence of isolated preovulatory follicles also showed augmented biphasic LH responses to GnRH compared with control hemi-pituitary glands. The apparent dissociation which can occur between follicular maturation, circulating oestrogen concentrations and pituitary responsiveness to GnRH supports the idea of non-steroidal ovarian factors modulating LH release.     

In vitro DNA synthesis by isolated preantral to preovulatory follicles from the cyclic mouse

In recent studies, we have shown that the smallest preantral follicles in the cyclic hamster increase DNA synthesis in the periovulatory period in response to surge levels of FSH. The current investigation was designed to determine whether the same phenomenon occurs in the cyclic mouse. Intact mouse follicles were isolated with watchmaker forceps (stages 4-6) or by enzymatic digestion (stages 1-4) at 0900 h and 1500 h on each day of the 5-day estrous cycle. The isolated follicles were classified into 6 stages: stages 1 and 2: follicles with 1 and 2 layers of granulosa cells; stage 3: follicles with 3 or more layers of granulosa cells and formation of theca; stages 4-6: incipient, small, and preovulatory antral follicles. The follicles at each stage were incubated for 3 h with [3H]thymidine. DNA content in stages 1-4 of follicles remained unchanged during the estrous cycle; for stages 5 and 6, DNA content was higher on the afternoon of proestrus than on other days of the cycle. Incorporation of [3H]thymidine for stages 1-3 (preantral follicles) started to increase at 1500 h of proestrus and peaked at 0900 h on estrus, whereas for stages 4-6, DNA synthesis peaked on proestrus (1500 h) and then fell by the morning of estrus. Thus, the rate of DNA replication in preantral and antral mouse follicles were different. Similarities and differences in folliculogenesis between mouse and hamster are discussed. These results suggest that DNA synthesis and the growth of all stages of follicles in the cyclic mouse may be associated with changing levels of periovulatory gonadotropins.     

Sequential changes associated with the degeneration of preovulatory rat follicles.

In 26-day-old rats, follicles capable of ovulation were present 48 h after PMSG injection and they degenerated if not exposed to an ovulating dose of HCG. In such follicles 125I-labelled LH bound to the thecal and granulosa cells. By 60 h after PMSG, LH binding to the granulosa cells was reduced by 46% although these follicles retained their ability to ovulate. LH binding to the granulosa cells was lost in most follicles by 72 h and ovulation could not be induced. The thecal cells still possessed LH binding sites at 72 h after PMSG. HCG stimulation of these follicles resulted in disruption of the granulosa and the invasion of blood cells into the antrum.     

GnRH actions on rat preovulatory follicles are mediated by paracrine EGF-like factors

Gonadotropin releasing hormone (GnRH) has been shown to mimic the actions of LH/hCG on oocyte maturation and ovulation. Recent studies demonstrated that induction of ovulation by LH/hCG is mediated, at least in part, by transactivation of epidermal growth factor receptors (EGFR) by autocrine/paracrine EGF-like factors activated by metalloproteases. Here we have examined whether the action of GnRH on the preovulatory follicles is exerted through similar mechanisms involving activation of EGFR. The EGFR kinase inhibitor, AG1478, inhibited GnRH-induced oocyte maturation in explanted follicles in vitro. Its inactive analog, AG43, did not affect GnRH-stimulated resumption of meiosis. GnRH, like LH, stimulated transient follicular expression of EGF-like agents, as well as rat cycloxygenase-2 (rCOX-2), rat hyaluronan synthase-2 (rHAS-2), and rat tumor necrosis factor-alpha-stimulated gene 6 (rTSG-6) mRNAs, known ovulatory enzymes. Likewise, GnRH stimulated follicular progesterone synthesis. Conversely AG1478 inhibited all these actions of GnRH. Furthermore, Galardin, a broad-spectrum metalloprotease inhibitor, blocked GnRH-induced oocyte maturation and follicular progesterone synthesis. In conclusion, we have demonstrated that follicular EGF-like factors mediate also the GnRH-stimulation of ovulatory changes, like these of LH/hCG.     

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.     

The role of protein kinase C in the stimulation of phosphatidylcholine synthesis by phospholipase C

The role of protein kinase C in the stimulation of phosphatidylcholine (PC) synthesis by phospholipase C was investigated. Phospholipase C treatment of Chinese hamster ovary cells (CHO) generates diacylglycerol, which is an activator of protein kinase C. The protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated choline incorporation into two CHO cell lines, a wild-type cell line, WTB, and a mutant cell line, DTG 1-5-4. DTG 1-5-4 is a mutant defective in receptor-mediated endocytosis. A 3-h phospholipase C treatment resulted in the activation and translocation of CTP:phosphocholine cytidylyltransferase in both cell lines. TPA treatment, however, resulted in only a slight (20%) translocation of cytidylyltransferase in WTB; no detectable translocation of cytidylyltransferase was observed in DTG 1-5-4. A decrease in the phosphocholine pools was observed in response to TPA treatment in both cell lines, which indicated that the cytidylyltransferase step was being activated. Phospholipase C stimulated choline incorporation into PC even when protein kinase C had been down-regulated in both cell lines. It was concluded that phospholipase C does not activate PC synthesis by activating protein kinase C.     

Effect of human chorionic gonadotrophin and indomethacin on ovulation, steroidogenesis and prostaglandin synthesis in preovulatory follicles of PMSG-primed immature rats

Immature rats were treated with PMSG followed 56 h later by 10 i.u. hCG. Follicles were removed at intervals after hCG injection. Transient increases in progesterone, testosterone and oestradiol synthesis were first evident 1 h after hCG, but values peaked at 3-5 h and returned to control levels by 10 h. Increased synthesis of PGE-2 and PGF-2 alpha was not evident until 3 h and peaked at more than 10 h after hCG. Ovulation began between 8 and 10 h after hCG and 83% of animals had ovulated within 12 h. Doses of 90 or 1800 micrograms indomethacin given together with hCG substantially inhibited ovulation and PG synthesis, but only the higher dose inhibited the hCG-induced elevation of progesterone and testosterone synthesis; hCG-induced oestradiol synthesis was not affected by either dose of indomethacin. We conclude that the peak of PG synthesis after hCG treatment related closely to the timing of ovulation; the steroidogenic response to hCG was not blocked by doses of indomethacin sufficient to inhibit synthesis of PGE-2 and PGF-2 alpha by more than 80%.