| 1. |
- Bicsak, T A, et al.
(författare)
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Tissue-type plasminogen activator in rat oocytes : expression during the periovulatory period, after fertilization, and during follicular atresia.
- 1989
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 124:1, s. 187-94
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Tsafriri, A, et al.
(författare)
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Suppression of ovulation rate by antibodies to tissue-type plasminogen activator and alpha 2-antiplasmin.
- 1989
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 124:1, s. 415-21
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Tidskriftsartikel (refereegranskat)abstract
- Indirect evidence has suggested a role for plasminogen activator (PA) in ovulation. Our recent studies demonstrated that 1) tissue-type PA (tPA) is the predominant PA produced by preovulatory rat follicles in response to gonadotropins or GnRH; and 2) several inhibitors of the serine proteases, to which PA and plasmin belong, block ovulation. Here, the role of tPA and plasmin in ovulation was examined directly by the use of specific antibodies to tPA and alpha 2-antiplasmin (alpha 2AP). Immature female rats at 25-26 days of age were treated (sc) with 15 IU PMSG to induce multiple preovulatory follicles. Fifty-four hours later, tPA antibodies and alpha 2AP were injected into one of the ovarian bursae to check their ability to block ovulation, which was initiated with an ovulatory dose (4 IU) of hCG. The data are expressed as percent inhibition of ovulation in the treated vs. the untreated ovaries. A significant decrease in the ovulation rate was obtained by administration of 500 micrograms antibodies to tPA (39.6%) or 1-50 micrograms alpha 2AP (36-44%), whereas minimal inhibition (12%) was found at lower doses of anti-tPA (10 micrograms) or alpha 2AP (0.1 micrograms). Furthermore, nonimmune immunoglobulin G (500 micrograms) and heat-inactivated alpha 2AP were not effective. Anti-tPA and alpha 2AP suppressed ovulation only when injected at the time of hCG administration; later injections (4-h delay) were ineffective, suggesting that PA and plasmin are involved in the early follicular responses to the ovulatory stimulus. Histological observation of the ovaries did not reveal any pathological changes associated with the anti-tPA and alpha 2AP treatment. Suppression of ovulation, as evidenced by decreased number of tubal ova, was frequently accompanied with intraovarian release of the eggs into the follicular thecal compartment. Thus, these results provide direct evidence for an essential role of tPA and plasmin in ovulation.
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| 3. |
- Galway, A B, et al.
(författare)
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Epidermal growth factor stimulates tissue plasminogen activator activity and messenger ribonucleic acid levels in cultured rat granulosa cells : mediation by pathways independent of protein kinases-A and -C.
- 1989
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 125:1, s. 126-35
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Tidskriftsartikel (refereegranskat)abstract
- Recent reports suggest that epidermal growth factor (EGF) or related peptides may act as local hormones to regulate granulosa cell differentiation. While FSH and GnRH are known to stimulate accumulation of tissue-type plasminogen activator (tPA) mRNA in granulosa cells, studies using nonovarian cells have shown stimulation of tPA by EGF. In this study, the effect of EGF and its structural analog transforming growth factor-alpha (TGF alpha) on ovarian tPA mRNA and activity was investigated. Granulosa cells obtained from immature estrogen-treated rats were cultured with FSH or increasing doses of EGF or TGF alpha before analysis of tPA activity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique. Like FSH and GnRH, EGF and TGF alpha stimulated the secretion of tPA activity in a dose- and time-dependent manner (onset, 12 h; maximum, 48 h). Northern blot hybridization of total RNA using a rat cRNA probe for tPA showed the accumulation of a 22S species mRNA in cells treated with EGF or TGF alpha, but not with nerve growth factor, suggesting increased expression of the tPA gene. Furthermore, slot blot hybridization of RNA from these cells confirmed a time-dependent increase in tPA mRNA preceding that in enzyme activity. Cotreatment of a saturating dose of EGF with phorbol myristate acetate (PMA) or GnRH resulted in additive increases in both tPA enzyme activity and mRNA levels. In addition, pretreatment with PMA desensitized the cells to subsequent treatment with PMA or GnRH, but did not diminish EGF-induced tPA mRNA, suggesting that EGF acts through a pathway independent of protein kinase-C. Also, extracellular cAMP levels did not increase with EGF treatment in the presence or absence of a phosphodiesterase inhibitor, suggesting the lack of involvement of the protein kinase-A pathway. Suppression of protein synthesis by cycloheximide inhibited the induction of tPA mRNA by EGF, whereas similar treatment resulted in the superinduction of tPA mRNA in FSH-treated cells, suggesting that EGF and FSH do not share the same pathway. These results suggest that EGF and TGF alpha induce tPA mRNA and activity in granulosa cells through a pathway independent of protein kinases-A (FSH) and -C (GnRH and phorbol ester), providing an interesting model for future elucidation of the molecular mechanism involved in tPA gene expression.
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| 4. |
- Ny, Tor, et al.
(författare)
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Cultured granulosa cells produce two plasminogen activators and an antiactivator, each regulated differently by gonadotropins.
- 1985
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 116:4, s. 1666-8
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Tidskriftsartikel (refereegranskat)abstract
- Although treatment of cultured granulosa cells with gonadotropins increases their fibrinolytic activity, the biochemical nature of this effect is unclear. We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fibrin autography techniques to characterize the fibrinolytic components secreted by granulosa cells. The fibrinolytic activity of these cells results from the production of both a tissue-type plasminogen activator (t-PA) and a urokinase-like activator (u-PA). The cells also produce an inhibitor of fibrinolysis (antiactivator). FSH and LH stimulate t-PA activity and suppress antiactivator activity, while u-PA activity is not affected by the gonadotropins. The differential regulation of these molecules by the gonadotropins may be essential for ovulation.
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| 5. |
- Hsueh, A J, et al.
(författare)
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Gonadotropin-releasing hormone induces ovulation in hypophysectomized rats : studies on ovarian tissue-type plasminogen activator activity, messenger ribonucleic acid content, and cellular localization.
- 1988
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 122:4, s. 1486-95
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 6. |
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| 7. |
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| 8. |
- Liu, Y X, et al.
(författare)
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Gonadotropin regulation of tissue-type and urokinase-type plasminogen activators in rat granulosa and theca-interstitial cells during the periovulatory period.
- 1987
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Ingår i: Molecular and Cellular Endocrinology. - 0303-7207 .- 1872-8057. ; 54:2-3, s. 221-9
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Tidskriftsartikel (refereegranskat)abstract
- 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)
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| 9. |
- Liu, Y X, et al.
(författare)
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Identification and regulation of tissue plasminogen activator activity in rat cumulus-oocyte complexes.
- 1986
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Ingår i: Endocrinology. - 0013-7227 .- 1945-7170. ; 119:4, s. 1578-87
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Tidskriftsartikel (refereegranskat)abstract
- Plasminogen activators convert plasminogen into plasmin, a serine protease that initiates extracellular proteolysis. Two types of plasminogen activator activities have recently been demonstrated in granulosa cells, and the proteolysis-inducing enzymes are believed to be involved in ovulation. However, little attention has been paid to the presence of these enzymes in oocytes. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by a fibrin overlay technique, we studied plasminogen activator activity in oocytes. Denuded oocytes collected from ovaries of hypophysectomized, estrogen-treated immature rats contained a tissue-type plasminogen activator (tPA), but not urokinase (uPA). In contrast, oocyte-free granulosa cells in these preantral follicles contained uPA, but not tPA. The tPA activity found in oocytes was plasminogen-dependent; incubation with increasing numbers (25-200) of denuded oocytes resulted in a dose-dependent increase in fibrinolysis only in the presence of plasminogen. Cellular localization of tPA was studied in the preantral follicles using an immuno-cytochemical method. Positive tPA staining was detected in the cytoplasm, but not in the germinal vesicle or zona pellucida of the oocytes. Furthermore, analysis using a reverse fibrin-overlay method did not reveal the presence of a plasminogen activator inhibitor. Culturing of denuded oocytes for 24 h increased the cellular content of tPA, but the enzyme activity was not further enhanced by treatment with FSH or forskolin. Also, no tPA activity was detected in the medium. We further studied plasminogen activator activities in the cumulus-oocyte complexes. Although only tPA activity was detected in freshly obtained cumulus-oocyte complexes, incubation for 24 h increased both tPA and uPA activity. Furthermore, tPA, but not uPA, activity was stimulated by treatment with FSH or forskolin. This was accompanied by the secretion of tPA into the medium. The identity of tPA and uPA in the cumulus-oocyte complexes was further confirmed by immunoprecipitation with specific antibodies. Isolation of denuded oocytes and cumulus cells after hormonal stimulation of the cumulus-oocyte complexes suggested that tPA activity was stimulated in both cell types and that the cumulus cells may mediate the action of FSH and forskolin on oocytes. In conclusion, the detection and regulation of tPA activity in cumulus-oocyte complexes suggest possible involvement of this enzyme in ovulation or the process of cumulus cell expansion and dispersion. Changes in oocyte tPA content may also serve as an indicator of oocyte development.
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| 10. |
- Ny, Tor, et al.
(författare)
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Cloning and characterization of a cDNA for rat tissue-type plasminogen activator.
- 1988
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Ingår i: DNA (Mary Ann Liebert, Inc.). - 0198-0238. ; 7:10, s. 671-7
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Tidskriftsartikel (refereegranskat)abstract
- Two partly overlapping lambda gt11 cDNA clones coding for the 22S rat tissue-type plasminogen activator (t-PA) mRNA were isolated. The cDNA sequences cover 2445 nucleotides of the mRNA, including a 5' untranslated region of 31 nucleotides, an open reading frame of 1677 nucleotides, a 3' untranslated region of 737 nucleotides, and a poly(A) tail. The open reading frame codes for a 17-amino-acid signal peptide, a propeptide with 12 amino acids, and the mature protein with 530 amino acids. Rat t-PA has 81% and 92% amino acid sequence identity with the human and mouse counterparts and an equal distribution of conserved amino acids, suggesting that the proteins can fold into identical three-dimensional structures. The rat t-PA sequence contains two putative N-glycosylation sites at Asn-120 and Asn-452, while human t-PA has an additional glycosylation site at Asn-187. The site at Asn-187 is glycosylated in the human protein, revealing a different glycosylation pattern between the human and rat proteins.
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