Tissue-type plasminogen activator and its inhibitor plasminogen activator inhibitor type 1 are coordinately expressed during ovulation in the rhesus monkey

Ovulation is a gonadotropin-controlled process that is essential for the propagation of all mammalian species. In the present study, we used a pregnant mare serum gonodotropin/human chorionic gonadotropin (hCG)-induced, synchronized ovulation model in rhesus monkeys and systematically investigated the roles of the plasminogen activator (PA) system in the ovulatory process of the primate. At different follicular developmental stages throughout the periovulatory period, samples of ovaries, granulosa cells, and theca-interstitial cells as well as follicular fluid were collected, and levels of PA and PA inhibitor type-1 (PAI-1) were evaluated by fibrin overlay, reverse fibrin overlay, Northern blot analysis, and in situ hybridization, respectively. We showed that in response to an injection of ovulation-triggering hCG, which mimics the preovulatory surge of LH in the circulation, granulosa cell-derived tissue-type PA (tPA) was substantially elevated in preovulatory follicles and reached its maximum level just before ovulation. Although theca-interstitial cell-derived PAI-1 was also stimulated by pregnant mare serum gonodotropin and hCG treatments, however, the maximum level of PAI-1 appeared 12 h earlier than that of tPA. When ovulation approached, accompanying the highest tPA level in the preovulatory follicles, the follicular PAI-1 level declined dramatically to its minimum value. Moreover, our data on the expression of follicular PA and PAI-1 over the periovulatory period were reinforced by results obtained at the mRNA level. Our data suggest that the coordinated expression of tPA and PAI-1 may be of importance for the follicular rupture process during ovulation in the primate.     

Concentrations of arachidonate metabolites, steroids and histamine in preovulatory horse follicles after administration of human chorionic gonadotrophin and the effect of intrafollicular injection of indomethacin

This study investigated the sequence of hormonal changes within the preovulatory follicles of mares. Mares were injected i.v. with 2500 IU human chorionic gonadotrophin (hCG) when a preovulatory follicle of 35 mm in diameter was detected. Fluid was aspirated from preovulatory follicles before (0 h), and 12, 24 and 36 h after administration of hCG. Concentrations of progesterone, prostaglandin (PG) E2, PGF, 6-keto-PGF1 alpha and thromboxane B2 in follicular fluid increased significantly (P less than 0.01) between 0 and 36 h. At 36 h, PGE2 was present in highest concentrations, followed by PGF and 6-keto-PGF1 alpha; thromboxane B2 was present at lower concentrations than other prostanoids. Concentrations of 13,14-dihydro-15-keto-PGF2 alpha increased significantly (P less than 0.05) between 24 and 36 h. Leukotriene B4, leukotriene C4 and histamine were present in follicular fluid at all sampling periods and did not change significantly over time. In another experiment, buffered saline or indomethacin (either 100 or 500 micrograms) was injected into preovulatory follicles on the day that they reached 35 mm in diameter to determine whether blocking intrafollicular PG synthesis would affect ovulation. The interval between intrafollicular injection and ultrasonographic detection of luteinization was significantly longer (P less than 0.05) in mares treated with 500 micrograms indomethacin. Plasma progesterone concentrations were significantly (P less than 0.05) lower in indomethacin-treated mares than in control mares on the first 5 days after injection. These results indicate that intrafollicular concentrations of PGs increase significantly before ovulation in mares and may be involved in the ovulatory process.     

Effect of prostaglandins on collagen synthesis in rabbit ovarian follicles during the ovulatory process

Collagen fibers in the ovarian follicle undergo drastic changes at ovulation due to the preovulatory increase of collagenolytic activities. The collagen synthesis in ovaries, however, has not been elucidated yet. To clarify the regulatory role of prostaglandins (PGs) in collagen synthesis of the follicular wall in relation to the ovulatory process, we measured prolyl hydroxylase (PH), as well as lysyl oxidase (LO) activity and the content of hydroxyproline (Hyp) in ovarian follicles of the rabbits treated by hCG, hCG/indomethacin (IM) and hCG/IM/various PGs. The experimental groups consisted of; 1) untreat control group 2) ovulatory group receiving hCG 3) non-ovulatory group given PGs 4) ovulatory group given hCG and PGs 5) group in which hCG-induced ovulation was inhibited by IM (4 mg/kg) 6) group in which IM-inhibited ovulation was recovered by PGF2 alpha (1.5 mg/kg) 7) group in which IM-inhibited ovulation was not restored by PGE1 (0.1 mg/kg) and PGE2 (0.7 mg/kg). The peak activities of PH and LO in ovarian follicles were observed at 12-13 hr after hCG injection, namely, immediately after ovulation. Significant changes of these activities after hCG administration were specific to the ovaries. PH activity in the ovaries was suppressed by the administration of IM, but LO activity was not significantly suppressed. In the hCG/IM/PGF2 alpha-treated ovulatory rabbits (Group 6), PH activity recovered to nearly the level of the hCG-treated rabbits (Group 2). By addition of PGE2, ovulation did not recover but PH activity was restored to about 70% of the hCG-treated rabbits. PGE1 did not have any effect on the reversal of ovulation-blockage or restoration of PH activity. The amount of Hyp after hCG administration tended to decrease from 6 hr to 10 hr but was significantly increased from 10 hr to 13 hr. This increase of Hyp after ovulation significantly correlated with the increase of PH and LO activities. In the hCG/IM/PGF2 alpha-treated rabbits (Group 6), the changes of Hyp were similar to those the hCG-treated rabbits (Group 2). In conclusion, collagen synthetic activity, found to be regulated by PH and LO activities in the ovarian follicles, was activated after follicle rupture, resulting in reconstruction of collagen fibers, and PGs play an important role in the ovulatory process by modifying collagen synthesis.     

Gonadotropin regulation of tissue-type and urokinase-type plasminogen activators in rat granulosa and theca-interstitial cells during the periovulatory period

Plasminogen activators (PAs) are believed to be involved in ovulation. Because both tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) are secreted by cultured rat granulosa cells, we have examined the activities of these proteins in ovarian homogenates as well as granulosa and theca-interstitial (TI) cells during gonadotropin-induced ovulation. Immature rats were injected with 20 IU pregnant mare serum gonadotropin (PMSG) to initiate follicle development, followed by treatment with 10 IU hCG 48 h later to induce ovulation. Ovarian proteins were separated by SDS-PAGE and PA activity determined by fibrin overlay. The activity of tPA, but not uPA, was stimulated following PMSG treatment in ovarian homogenates. Subsequent hCG injection further increased the tPA activity in a time-dependent manner, reaching a maximum (12 h after hCG treatment) immediately prior to ovulation and declined thereafter. Similar preovulatory increases in tPA activity were detected in isolated granulosa cells. Although both tPA and uPA activities were increased in TI cells after PMSG administration, no further increases were detected after hCG treatment. To estimate enzyme secretion, ovarian cells obtained at various preovulatory periods were incubated for 24 h in vitro. The ability of granulosa cells to secrete tPA, but not uPA, increased following in vivo PMSG and hCG treatment in a time-dependent manner, reaching a maximum immediately prior to ovulation. During the preovulatory period, an abrupt increase in tPA secretion by TI cells was also detected. Using immunohistochemical staining for tPA, it was found that ovarian sections from preovulatory rats at 12 h after hCG injection stained positively in granulosa, theca interna, and interstitial gland cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin E2 acts via multiple receptors to regulate plasminogen-dependent proteolysis in the primate periovulatory follicle

The ovulatory gonadotropin surge regulates expression of plasminogen activator (PA) family members within the ovarian follicle, which are implicated in follicle wall degradation at ovulation. Gonadotropin also stimulates follicular prostaglandin E2 (PGE2) production, which is required for follicle rupture. To determine whether the ovulatory gonadotropin surge regulates PA-mediated proteolysis via PGE2 in the primate follicle, monkeys received gonadotropins to stimulate follicle development. Follicular aspirates or whole ovaries were obtained before (0 h) and after human chorionic gonadotropin (hCG) administration to span the periovulatory interval. Granulosa cell levels of tissue-type PA (tPA) and PA inhibitor type 1 (PAI-1) proteins were low at 0 h hCG and higher after hCG administration. In situ zymography showed no ovarian tPA activity 0 h after hCG; tPA activity was present in granulosa cells obtained after hCG treatment. Importantly, tPA and PAI-1 proteins and tPA activity were low/nondetectable in granulosa cells obtained after treatment with hCG and the PG synthesis inhibitor celecoxib. To determine whether hCG stimulation of tPA and PAI-1 requires PGE2, granulosa cells obtained at 0 h were cultured with hCG plus indomethacin to inhibit PG production; some cells also received PGE2 or an agonist selective for one PGE2 receptor (EP). PGE2, an EP2 agonist, and an EP3 agonist increased tPA protein, whereas PGE2, an EP1 agonist, and an EP3 agonist increased PAI-1 protein. Therefore, gonadotropin increases granulosa cell tPA and PAI-1 protein levels and tPA-dependent proteolytic activity. PGE2 also increases tPA and PAI-1 protein levels in granulosa cells, suggesting that elevated PGE2 late in the periovulatory interval acts to stimulate proteolysis and follicle rupture.     

Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats: studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization

GnRH and its agonists are known to induce ovulation in hypophysectomized rats by acting directly at the ovary. Because tissue-type plasminogen activator (tPA) has been implicated in the gonadotropin induction of ovulation, we examined the effect of an ovulatory dose of GnRH on ovarian tPA activity, mRNA content, and cellular localization. Hypophysectomized immature rats were injected sc with 20 IU PMSG and a single dose of a GnRH agonist (GnRHa; des-Gly10,DLeu6(N alpha Me)Leu7,Pro9NHEt-GnRH) 58 h later. At different times after treatment, ovaries were prepared for morphological analysis. Using a fibrin overlay method, tPA activities were measured in ovarian homogenates and cumulus-oocyte complexes, whereas granulosa cells were cultured for 24 h to estimate tPA secretion. Total ovarian RNA was prepared for hybridization analysis of tPA message levels, and tPA localization was studied by immunohistochemistry of ovarian sections. GnRHa induced ovulation in PMSG-primed hypophysectomized rats 14-16 h after injection in a dose-dependent manner, and the GnRHa action was blocked by concomitant treatment with a GnRH antagonist. GnRHa stimulated the induction of tPA, but not urokinase-type PA, activity in ovarian homogenates and granulosa cell-conditioned medium in a time-dependent manner, reaching a maximum before ovulation. tPA activity in cumulus-oocyte complexes was also increased before ovulation, but this increase was sustained. Hybridization analysis of steady state tPA mRNA levels was performed using a rat cRNA probe. Northern blot analysis of total ovarian RNA demonstrated that GnRHa stimulated tPA mRNA levels 12 h after treatment, with a subsequent decrease 24 h after treatment. Immunohistochemistry indicated substantial increases in tPA staining in granulosa cells and oocytes of preovulatory follicles before ovulation. Thus, GnRHa acts through specific receptors to increase ovarian tPA enzyme activity, mRNA content, as well as immunostaining in granulosa cells and oocytes. Like gonadotropins, GnRH may induce ovulation by directly stimulating tPA levels in the ovary.     

Reduced ovarian follicular development as a consequence of low body condition in ewes

The effect of body condition on ovarian follicular development was investigated in Scottish Blackface ewes in high and low body condition. Follicles were dissected from ovaries on days 11 and 12 of the luteal phase and 24 h after prostaglandin-induced luteal regression. Ewes in low body condition had a lower ovulation rate (low: 0.9; high: 1.8 P less than 0.05) and lower mean plasma levels of FSH during both the luteal (low: 54; high: 72 micrograms/l) and follicular (low: 34; high: 43 micrograms/l) phases of the cycle. Low body condition was associated with a reduced number of large (greater than or equal to 4 mm) follicles in both the luteal and follicular phases, and in low condition a lower proportion of these follicles was oestrogenic and potentially ovulatory as assessed by follicular fluid levels of oestradiol. However, within the different oestrogenic classifications of these large follicles there were no significant differences in the steroidogenic capacity as assessed by the concentrations of either oestradiol or testosterone in follicular fluid, basal and hCG-stimulated testosterone production, thecal 125I hCG binding or basal and testosterone-stimulated oestradiol production by granulosa cells in relation to body condition. These results suggest that body condition influences ovulation rate by altering the concentration of FSH in blood, which in turn affects the number of potentially ovulatory follicles growing beyond 4 mm.     

Alterations in nitrate/nitrite and nitric oxide synthase in preovulatory follicles in gonadotropin-primed immature rat

Nitric oxide synthase (NOS) and nitric oxide (NO) play important roles in ovulation. The purpose of this study was to investigate the changes of intrafollicular nitrate/nitrite concentration and NOS mRNA expression in preovulatory follicles during equine CG (eCG) and human CG (hCG) induced ovulation in immature rats. Immature Sprague-Dawley rats received 15 IU eCG and then 15 IU hCG 48 h later. Rats were killed immediately before, 5 h after or 10 h after hCG injection, and their preovulatory follicles were dissected. Follicular fluid, granulosa cell, and theca cell layers were collected from preovulatory follicles and assayed for NO or NOS mRNA or for in vitro incubation study. Nitrate/nitrite concentration in the follicular fluid decreased significantly 5 and 10 h after hCG injection. Inducible NOS (iNOS) mRNA expression, which was greater in granulosa cell than in the theca cell layer, decreased significantly 5 and 10 h after hCG injection. However, endothelial NOS (eNOS) mRNA expression was detected mainly in the theca cell layer and further increased 5 and 10 h after hCG injection but remained low in granulosa cells. In vitro treatment of granulosa cells with 10(-4) or 5x10(-4) M S-nitroso-L-acetyl penicillamine (NO donor) decreased progesterone production and increased DNA fragmentation. We concluded that the decrease in nitrate/nitrite concentration in preovulatory follicles after hCG injection was due mainly to decreased iNOS expression in granulosa cells. These changes in nitrate/nitrite concentration may prevent apoptosis in preovulatory follicles.     

Ovulation efficiency is reduced in mice that lack plasminogen activator gene function: functional redundancy among physiological plasminogen activators.

Several lines of indirect evidence suggest that plasminogen activation plays a crucial role in degradation of the follicular wall during ovulation. However, single-deficient mice lacking tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), or PA inhibitor type 1(PAI-1) gene function were recently found to have normal reproduction, although mice with a combined deficiency of tPA and uPA were significantly less fertile. To investigate whether the reduced fertility of mice lacking PA gene function is due to a reduced ovulation mechanism, we have determined the ovulation efficiency in 25-day-old mice during gonadotropin-induced ovulation. Our results reveal that ovulation efficiency is normal in mice with a single deficiency of tPA or uPA but reduced by 26% in mice lacking both physiological PAs. This result suggests that plasminogen activation plays a role in ovulatory response, although neither tPA nor uPA individually or in combination is obligatory for ovulation. The loss of an individual PA seems to be functionally complemented by the remaining PA but this compensation does not appear to involve any compensatory up-regulation. Our data imply that a functionally redundant mechanism for plasmin formation operates during gonadotropin-induced ovulation and that PAs together with other proteases generate the proteolytic activity required for follicular wall degradation.     

Ovarian responses of pregnant mare serum gonadotropin- and human chorionic gondotropin-primed rats: desensitizing, luteolytic, and ovulatory effects of a single dose of human chorionic gonadotropin.

We conducted a study to determine the morphological appearance and functional responsiveness of ovarian tissues after administration of hCG to 28-day-old rats primed 65 h earlier with PMS gonadotropin (PMSG) and after administration of a second dose of hCG 5 days later, i.e. to 33-day-old rats containing heavily luteinized ovaries. Sixty-five hours after the administration of 50 IU PMSG sc to 25-day-old rats, ovaries already contained an abundance of luteinized follicles and an adenylyl cyclase (AC) system that was responsive to LH, epinephrine, and NaF. The administration of 50 IU hCG sc at this time initially resulted in a loss of LH-responsive ovarian AC. Within 4 days of the hCG injection, the ovaries of the now 32-day-old rats were heavily luteinized, and ovarian AC was highly responsive to LH, epinephrine, and NaF. The administration of a single sc dose of 200 IU hCG to 33-day-old PMSC- and hCG-primed rats with luteinized ovaries resulted in a rapid desensitization of the ovarian AC to LH and a drop in serum progesterone levels, During the subsequent 7 days, serum progesterone levels continued to decline, while total ovarian AC reacquired responsiveness to LH by days 4--5 after the densensitizing dose of hCG. Dissection of ovarian components revealed, however, that the AC system of the corpora lutea originally present at the time of the second hCG injection remained permanently refractory to LH and that the AC in corpora lutea newly formed from freshly ovulated follicles exhibited a significant responsiveness to LH, epinephrine, and NaF. However, these new corpora lutea were not fully active, since serum progesterone never rose. Subcutaneous administration of 50 IU hCG to 33-day-old PMSG- and hCG-primed rats also promoted a rapid loss of AC responsiveness to LH. This lower concentration of hCG was not sufficient to promote follicular development or ovulation, and the ovarian AC remained refractory to LH for at least 7 days. Intravenous administration of 75 IU hCG to 33-day-old PMSG- and hCG-primed rats similarly promoted a rapid and permanent loss of luteal AC responsiveness to LH; again, follicles did not mature to a preovulatory state and, in fact, appeared to undergo atresia rather than ovulation. These results indicate that in heavily luteinized ovaries 1) hCG promotes desensitization of rat luteal AC to LH, 2) Desensitization of AC to LH stimulation in corpora lutea is permanent and irreversible, and 3) only under conditions where follicles mature and ovulate and new corpora lutea are formed does total ovarian AC reacqure responsiveness during the subsequent week.     

Midcycle administration of a progesterone synthesis inhibitor prevents ovulation in primates.

Progesterone receptors appear in granuloma cells of preovulatory follicles after the midcycle gonadotropin surge, suggesting important local actions of progesterone during ovulation in primates. Steroid reduction and replacement during the gonadotropin surge in macaques was used to evaluate the role of progesterone in the ovulatory process. Animals received gonadotropins to induce development of multiple preovulatory follicles, followed by human chorionic gonadotropin (hCG) administration (day 0) to promote oocyte (nuclear) maturation, ovulation, and follicular luteinization. On days 0-2, animals received no further treatment; a steroid synthesis inhibitor, trilostane (TRL); TRL + R5020; or TRL + dihydrotestosterone propionate (DHT). On day 3, ovulation was confirmed by counting ovulation sites and collecting oviductal oocytes. The meiotic status of oviductal and remaining follicular oocytes was evaluated. Peak serum estradiol levels, the total number of large follicles, and baseline serum progesterone levels at the time of hCG administration were similar in all animals. Ovulation sites and oviductal oocytes were routinely observed in controls. Ovulation was abolished in TRL. Progestin, but not androgen, replacement restored ovulation. Relative to controls, progesterone production was impaired for the first 6 days post-hCG in TRL, TRL + R5020, and TRL + DHT. Thereafter, progesterone remained low in TRL but recovered to control levels with progestin and androgen replacement. Similar percentages of mature (metaphase II) oocytes were collected among groups. Thus, steroid reduction during the gonadotropin surge inhibited ovulation and luteinization, but not reinitiation of oocyte meiotic maturation, in the primate follicle. The data are consistent with a local receptor-mediated role for progesterone in the ovulatory process.     

The involvement of collagenolysis in ovulation in the rat

Collagenolytic activity in ovarian follicles was previously demonstrated by using synthetic peptides and reconstituted collagen fibers. However, attempts to demonstrate degradation of ovarian collagen and to correlate collagenase activity with ovulation were not successful. By administration of L-(5-3H) proline, we have labeled ovarian and follicular collagen and followed collagenolytic activity by separation of 3H-hydroxyproline (3H-Hyp) from acid hydrolyzates of ovarian tissue by HPLC. The level of ovarian and follicular 3H-Hyp decreased by about 40% on the afternoon of proestrus or after exogenous stimulation of ovulation by human CG (hCG), and this decrease was abolished by blocking the surge of gonadotropins with Nembutal. To verify that the observed reduction in 3H-Hyp was due to the action of a typical collagenase, the collagenous fraction was prepared from ovarian tissue and from preovulatory follicles before and after the ovulatory stimulus. The extracts were treated with trypsin (25 min, 25 C, 0.01 mg/ml) plasmin and p-amino-phenyl-mercuric acetate to fully activate the collagenase extracted along with collagen. Both, enzymatic and chemical activation of collagenase in vitro resulted in degradation of collagen. This degradation could be inhibited by cysteine and EDTA; both are classic inhibitors of mammalian collagenases. The activity of ovarian collagenase increased within 3 h after hCG-stimulation, peaked at 5-fold 6 h after hCG, and declined afterwards. Administration of cysteine (0.001-0.01 mmol) into the bursal cavity of proestrous rats blocked ovulation and breakdown of ovarian collagen in a dose-dependent manner. Cysteine effectively inhibited ovulation even when injected 7 h after the hCG stimulus. Inhibitors of arachidonic acid metabolism prevent ovulation. Indomethacin (inhibitor of cyclooxygenase) and nordihydroguaiaretic acid (inhibitor of lipoxygenase) blocked ovulation and inhibited hCG-induced ovarian collagenolysis. Collectively, these results corroborate the essential role of collagenolysis in follicular rupture in mammals.     

Prolactin inhibits plasminogen activator activity in the preovulatory follicles

The present study was designed to determine the effects of PRL on changes in morphology and plasminogen activator (PA) activity in the preovulatory follicles. Rabbit ovaries were perfused with hCG alone or with hCG plus at 10, 10(2), or 10(3) ng/ml. PRL at 10(3) ng/ml directly inhibited the degeneration and decomposition of surface epithelial cells induced by hCG exposure. The subsurface connective tissue was visualized by treatment with sodium dodecyl sulfate, which removed surface epithelial cells from the ovary, thereby exposing collagen fibrils and the basal lamina. Sodium dodecyl sulfate treatment revealed inhibition of connective tissue disruption at the apex of the follicle wall in PRL-treated ovaries. PA activity in mature follicles in perfused rabbit ovaries exposed to hCG increased from 1.40 +/- 0.08 to 28.4 +/- 4.25 IU/g tissue after 4 h of perfusion. The addition of PRL to the perfusate inhibited the hCG-stimulated increase in intrafollicular PA activity in a dose-dependent fashion. Although at 7 h mature follicles treated by hCG alone showed greater intrafollicular PA activity than those treated with hCG plus PRL, this difference was not significant. These results suggest that PRL may act directly by interfering with mechanical events within the ovary that are required for the rupture of mature Graafian follicles, probably via the inhibition of intrafollicular tissue PA activity.     

Tissue-type plasminogen activator in rat oocytes: expression during the periovulatory period, after fertilization, and during follicular atresia

The regulation of tissue-type plasminogen activator (tPA) in rat oocytes during the periovulatory period, in early embryos, and in oocytes during induced follicular atresia was studied using a quantitative chromogenic substrate assay. Oocytes and early embryos were collected from three ovulation models: 1) intact immature female rats treated with PMSG, followed by hCG 48 h later; 2) hypophysectomized immature rats treated with PMSG, followed by a GnRH agonist (GnRHa) 56 h later; and 3) adult cyclic rats on the mornings of proestrus and estrus and up to 5 days after fertilization. In addition, follicular atresia was induced by either withdrawal of diethylstilbestrol (DES) for 2 days or injection of GnRHa for 2 days in hypophysectomized DES-implanted immature rats. Treatment with PMSG alone did not increase oocyte tPA content (5-20 microIU/oocyte) in either immature rat model, but treatment with either hCG or GnRHa induced meiotic maturation and ovulation and increased tPA activity to 80 and 140 microIU/oocyte 24 h after hCG and GnRHa treatment, respectively. Northern blot analysis of total RNA extracted from oocytes of PMSG-treated rats indicated the presence of a specific tPA message at 22S. tPA levels were low in preovulatory oocytes obtained on proestrus morning and increased in ovulated oocytes on estrus morning. After fertilization, tPA levels remained high in the embryos on days 1-4 of pregnancy, but dropped dramatically on day 5. Furthermore, oocytes from atretic follicles of hypophysectomized DES-implanted rats after either DES withdrawal or GnRHa treatment contained elevated levels of tPA, coincident with germinal vesicle breakdown (GVBD). Immunohistochemical staining revealed tPA antigen only in those oocytes that had undergone apparent meiotic maturation, as confirmed by GVBD. Thus, oocytes contain tPA mRNA and synthesize the active protease under a variety of stimuli which result in GVBD. The observed periovulatory increase in oocyte tPA activity, its maintenance until day 5 of pregnancy, and expression of tPA in nonovulatory oocytes of atretic follicles suggest diverse functions for the oocyte and embryo tPA.     

Follicular plasminogen activator: involvement in ovulation

Production of plasminogen activator (PA) by granulosa cells (GC) and its stimulation by gonadotropins led to the suggestion that PA is involved in ovulation. However, whereas only LH may be regarded as the ovulation-inducing hormone in the rat, FSH was found to be much more potent than LH in enhancing PA production by GC. Assuming that the entire follicular wall, rather than isolated GC, is involved in follicular rupture, we have examined activity of PA in intact follicles. LH (NIH-LH-S23) was 5-fold more potent than FSH (NIH-FSH-S14), and purified ovine LH and FSH were equally potent in enhancing follicular PA activity. Furthermore, injection into the ovarian bursa of proestrous rats of epsilon-amino-caproic acid and benzamidine (0.05-0.25 mmol), inhibitors of serine proteases, including PA and plasmin, resulted in a dose-dependent inhibition of ovulation without causing changes discernible by histological examinations of the ovaries. Whereas steroids did not change basal follicular PA production in culture, addition of estradiol-17 beta [(E2) 1 microgram/ml] but not progesterone or testosterone, further enhanced LH-stimulated PA. Aminoglutethimide phosphate (10(-3) M) and 17 beta-formamidoandrost-4-en-3-one inhibited LH-induced increase in follicular PA and this inhibition was reversed by addition of E2. Intrabursal injection of indomethacin, an inhibitor of cyclooxygenase, and of nordihydroguaiaretic acid, an inhibitor of lipoxygenase pathway of arachidonic acid metabolism at doses which effectively blocked ovulation (0.3 mg/bursa) had no effect on PA content of the follicles. Likewise, indomethacin (10 microM) and nordihydroguaiaretic acid (100 microM) did not affect LH-stimulated PA in vitro. In conclusion, LH, the physiological trigger of ovulation is, at least, as potent as FSH in stimulating follicular PA activity. The role of serine proteases, most probably of PA and plasmin, in ovulation is further corroborated by a pharmacological approach. LH stimulation of follicular PA appears to be enhanced by E2 but is not mediated by arachidonic acid metabolites.     

Inhibition of first ovulation: administration of an LHRH antagonist to immature female rats

Subcutaneous injections of an LHRH antagonist (ALHRH; Org.30093) were administered to immature female rats. Neither a single high dose (50 micrograms) nor repeated daily doses of 5-30 micrograms ALHRH/day, administered between 28 and 38 days of age, influenced the age and body weight at the time of vaginal opening or first ovulation. If repeated daily doses of 2 X 10 micrograms ALHRH were given from 32 to 42 or from 37 to 47 days of age, first ovulation was delayed by 3.0 and 6.3 days respectively. Administration of 10 micrograms ALHRH at 09.00 h and again at 17.00 h on the day of first pro-oestrus was found to be sufficient to block the expected first ovulation in 36 out of 38 rats. This effect could be repeated by administering the same doses of ALHRH at pro-oestrus and again on the next day: ovulation was blocked in eight out of eight rats. A single dose of 10 micrograms ALHRH, administered on the morning of pro-oestrus, blocked ovulation in five out of twelve rats. Both the preovulatory LH and FSH surge, as measured at 16.00 h on pro-oestrus, were found to be inhibited by ALHRH treatment. On the day after pro-oestrus no recruitment of new small antral follicles had occurred in rats with ovulatory blockade. Delayed ovulation took place 2-5 days after ALHRH injection at pro-oestrus; until 3 days after injection rats were able to ovulate their original preovulatory follicles, thereafter newly developed follicles ovulated and large ovarian cysts were found in the ovaries, next to fresh corpora lutea.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of danazol on steroidogenesis and gonadotropic responsiveness in isolated human preovulatory follicular cells

In order to investigate the influence of danazol on steroidogenesis and gonadotropic responsiveness of human follicular cells, granulosa and thecal cells of preovulatory follicles were isolated and separately incubated for short term periods. Human chorionic gonadotropin (hCG) (100 IU/ml), FSH (0.5 IU/ml) and danazol (10 micrograms/ml) alone or in combination were added to the incubation medium. Following incubation the cellular cyclic adenosine 3'5' monophosphate (cAMP) levels and the medium content of progesterone (P), androstenedione (A) and 17 beta-estradiol (E2) were determined. All follicles included in the study were classified as nonatretic and well developed, i.e. less than 3 days before ovulation. Human chorionic gonadotropin caused an increase in cAMP formation in both cell-types and this effect was significantly counteracted by danazol in vitro. In granulosa cells danazol tended to counteract a stimulatory effect of FSH on cAMP formation. No significant influence of danazol was found on the basal steroid formation of both cell types during short term incubation. On the other hand, danazol significantly counteracted the FSH stimulated P formation of the granulosa cells and the hCG stimulated A and E2 formation of the thecal cells. It is concluded that danazol inhibits gonadotropin-stimulated steroidogenesis locally in the human follicular cells and that this effect of danazol is mediated via the cyclic AMP system.     

The role of protein kinase-C in gonadotropin-induced ovulation in the in vitro perfused rabbit ovary.

Tumor-promoting phorbol esters are believed to affect ovarian granulosa cell progesterone and prostaglandin (PG) production and possibly ovulation by activating protein kinase-C (PKC). The effects of phorbol esters and PKC inhibitors on ovulation, progesterone, and PG production were examined in an in vitro perfused rabbit ovary. The effect of tranexamic acid, an inhibitor of the conversion of plasminogen activator to plasmin, on phorbol ester-induced ovulation was also examined. Phorbol 12,13-dibutyrate (PdBU), a PKC stimulator, induced ovulation in a dose-related manner in the absence of gonadotropins (56%, 200 nM PdBU; 0%, 0 nM PdBU; P < 0.05). Perfusate progesterone levels were increased only after 600 nM PdBU treatment, and perfusate PGF2 alpha, PGE2, and 6-keto-PGF1 alpha were increased in a dose-dependent fashion (P < 0.05). Staurosporine, a potent inhibitor of the catalytic domain of PKC, and calphostin-C, a specific inhibitor of the diacylglycerol-binding region, inhibited hCG-induced ovulation in a dose-related manner. Gonadotropin-induced ovulation decreased from 73% without staurosporine to 19% with 1.0 microM staurosporine (P < 0.01). Calphostin-C reduced ovulatory efficiency from 60% to 24% (P < 0.01). However, neither inhibitor decreased progesterone or PGF2 alpha production by ovaries exposed to hCG. hCG-induced oocyte maturation was also unaffected by exposure to either staurosporine or calphostin-C. Tranexamic acid reduced phorbol ester-induced ovulatory efficiency from 67% to 37% (P < 0.05). These findings demonstrate that the calcium-dependent PKC pathway is instrumental in gonadotropin-mediated follicular rupture in the rabbit. Although PGs may play an important role in ovulation, they do not appear to be directly responsible for PKC-mediated follicular rupture.     

Hormonal regulation and tissue-specific localization of alpha 2-macroglobulin in rat ovarian follicles and corpora lutea

alpha 2-Macroglobulin (alpha 2M) is a broad spectrum protease inhibitor associated with inflammatory responses and proposed to be important in tissue remodeling. alpha 2 M also functions as a carrier of specific growth factors and cytokines, including platelet-derived growth factor, transforming growth factor-beta, basic fibroblast growth factor, interleukin-1, interleukin-6. To determine whether alpha 2M is associated with remodeling phenomena in the rat ovary, the expression of alpha 2M mRNA and protein has been analyzed in specific ovarian cell types during ovulation, luteinization, and luteolysis. Before ovulation, alpha 2M mRNA is not detectable in granulosa cells. Twelve hours after injection of an ovulatory dose of hCG a 5.2-kilobase alpha 2M mRNA is detectable in luteinizing follicles, which is increased further by 48 h and maintained in corpora lutea (CL) for up to 96 h. Administration of PRL from 24-96 h results in both inhibition of luteolysis and marked increases in alpha 2M mRNA in CL, but not in residual tissues, of these same ovaries, isolated 48, 72, and 96 h after an ovulatory dose of hCG, alpha 2M mRNA is also induced by PRL in cultures of luteinized granulosa cells. These changes in alpha 2M mRNA in follicles or developing CL do not appear to reflect the amount of alpha 2M protein present: alpha 2M protein (188K monomer) is present (immunoblot and immunofluorescence data) in small antral and preovulatory follicles even though mRNA is not detectable; after an ovulatory dose of hCG the protein level transiently increases by 12 h (approximately 5-fold) and declines thereafter through 96 h; the decrease in alpha 2M protein observed at 48-96 h is delayed but not abolished by treatment with PRL, even though the mRNA levels continue to rise during this same time period. In contrast, changes in alpha 2M mRNA and protein are regulated coordinately in CL of pregnant rats. alpha 2M mRNA is present, but in low concentration, from days 4-11 of gestation, increases markedly between days 11-21, and decreases at parturition, when functional luteolysis occurs. Hysterectomy of day 10 pregnant rats combined with hormone replacement determined that alpha 2M mRNA levels are regulated primarily by PRL through day 12 and by placental lactogens during midgestation (days 12-15). The increase in alpha 2M mRNA during pregnancy precedes the 40-fold increase (peak) in a alpha 2M protein observed on day 15, which remains elevated through day 21.(ABSTRACT TRUNCATED AT 400 WORDS)