Mouse ovarian granulosa cells produce urokinase-type plasminogen activator, whereas the corresponding rat cells produce tissue-type plasminogen activator

It is well established that rat ovarian granulosa cells produce tissue plasminogen activator (tPA). The synthesis and secretion of the enzyme are induced by gonadotropins, and correlate well with the time of follicular rupture in vivo. We have found that in contrast, mouse granulosa cells produce a different form of plasminogen activator, the urokinase-type (uPA). As with tPA synthesis in the rat, uPA production by mouse granulosa cells is induced by gonadotropins, dibutyryl cAMP, and prostaglandin E2. However, dexamethasone, a drug which has no effect on tPA synthesis in rat cells inhibits uPA synthesis in the mouse. Results of these determinations made in cell culture were corroborated by examining follicular fluid, which is secreted in vivo predominantly by granulosa cells, from stimulated rat and mouse ovarian follicles. Rat follicular fluid contained only tPA, and mouse follicular fluid only uPA, indicating that in vivo, granulosa cells from the two species are secreting different enzymes. The difference in the type of plasminogen activator produced by the rat and mouse granulosa cells was confirmed at the messenger RNA level. After hormone stimulation, only tPA mRNA was present in rat cells, whereas only uPA mRNA was found in mouse cells. Furthermore, the regulation of uPA levels in mouse cells occurs via transient modulation of steady-state levels of mRNA, a pattern similar to that seen with tPA in rat cells.     

Regulation of tissue-type plasminogen activator activity and messenger RNA levels by gonadotropin-releasing hormone in cultured rat granulosa cells and cumulus-oocyte complexes

Gonadotropin-releasing hormone (GnRH) acts directly on the ovary to induce ovulation in hypophysectomized proestrous rats. Because plasminogen activators (PAs) are implicated in gonadotropin-induced ovulation, we have studied the effect of GnRH on ovarian PA synthesis. GnRH induced tissue-type PA (tPA) secretion by cultured rat granulosa cells, but inhibited the secretion of urokinase-type PA. These effects were blocked by co-treatment with a GnRH antagonist, suggesting that stereospecific GnRH receptors are involved. Follicle-stimulating hormone (FSH) also induced tPA in granulosa cells but with a different time course than GnRH; the combined effect of FSH and GnRH was additive. The GnRH effect was mimicked by the calcium- and phospholipid-dependent protein kinase C activator, phorbol myristate acetate. In isolated cumulus-oocyte complexes and cumulus cells, GnRH treatment also increased tPA activity. In contrast, treatment of denuded oocytes with GnRH did not increase enzyme activity. After GnRH stimulation of the cumulus-oocyte complexes, tPA content in the denuded oocyte was elevated, suggesting that the cumulus cells mediate the action of GnRH to increase the oocyte enzyme levels. Hybridization experiments using a labeled rat tPA-specific DNA probe showed that both FSH and GnRH increased the level of tPA mRNA in cultured granulosa cells; the stimulatory effect of GnRH was blocked by the GnRH antagonist. Our results indicate that GnRH treatment increases tPA secretion by cultured granulosa cells and cumulus-oocyte complexes. The stimulation of enzyme activity in the granulosa cells is accompanied by increases in tPA mRNA levels.     

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.     

Tissue-specific and time-coordinated hormone regulation of plasminogen-activator-inhibitor type I and tissue-type plasminogen activator in the rat ovary during gonadotropin-induced ovulation

The plasminogen-activator system provides proteolytic activity in many biological processes. The regulation of plasminogen activation may occur at many levels including the synthesis and secretion of plasminogen activators (PA) and the specific inhibition of PA activity by inhibitors. PA-inhibitor type-1 (PAI-1) is an efficient inhibitor of tissue-type PA (tPA) and urokinase-type PA (uPA) that may therefore be instrumental for the control of plasminogen activation. To investigate if coordinated regulation of PA and PA inhibitors take place in vivo in response to physiological signals, we have examined the regulation of PAI-1 and tPA in the ovary during gonadotropin-induced ovulation. We found that PAI-1, as well as tPA activity and mRNA levels, were coordinately regulated by gonadotropins in a time-dependent and cell-specific manner, such that a surge of PA-activity was obtained just prior to ovulation. Both theca-interstitial and granulosa cells synthesized PAI-1, but their maximal PAI-1 expression occurred at different times during the periovulatory period, ensuring inhibition of proteolytic activity in ovarian extra cellular compartments both before and after ovulation. The coordinated regulation of tPA and PAI-1 in the ovary may fine-tune the peak of PA activity which may be important for the regulation of the ovulatory process.     

Pituitary adenylate cyclase-activating polypeptide modulates plasminogen activator expression in rat granulosa cell

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide isolated from ovine hypothalamus. It has been demonstrated to be transiently expressed in preovulatory follicles and to positively affect several parameters correlated with the ovulatory process. The aim of the present study was to investigate whether PACAP influences the plasminogen/plasmin system in rat ovary. Plasminogen activators (PAs) are serine proteases, modulated by gonadotropins and several peptides in preovulatory follicles, that appear to be involved in ovulation. Granulosa cells obtained from immature eCG-treated rats were cultured for 24 h in the presence of increasing concentrations of PACAP and vasoactive intestinal peptide (VIP). A significant, dose-dependent increase in tissue-type PA (tPA) activity and decrease in urokinase-type (uPA) PA activity were observed in PACAP-treated cells. These effects were exerted at the mRNA level. The use of cycloheximide, a protein synthesis inhibitor, suggested that PACAP requires an intermediary protein to decrease uPA-mRNA, but not to induce tPA-mRNA. However, no significant modulation of PAs was observed in the presence of VIP. When granulosa cells were stimulated within the intact follicle (i.e., maintaining the three-dimensional structure and in the presence of the theca cell layers), both PACAP and VIP dose-dependently stimulated tPA. These data suggest that, in addition to the PACAP type I receptor present on granulosa cells, different subtypes of PACAP receptors are present in the different ovarian compartments.     

Plasminogen activator activity in cumulus-oocyte complexes of gonadotropin-treated rats during the periovulatory period

Two types of plasminogen activator (tissue-type, tPA; urokinase-type, uPA) have been demonstrated in ovarian granulosa cells, but only tPA activity was found in denuded oocytes. Immature rats were treated subcutaneously with 20 IU pregnant mare's serum gonadotropin (PMSG) to stimulate follicle maturation, followed 2 days later by an injection of 10 IU human chorionic gonadotropin (hCG) to induce ovulation. Cellular plasminogen activator activities were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis followed by a fibrin-overlay technique. Cumulus-oocyte complexes from rats before and after PMSG treatment contained low amounts of tPA, but not uPA, activity. After hCG treatment, tPA activity showed a time-dependent increase, reaching a maximum at 24 h after injection. At 12 and 24 h after hCG treatment, uPA activity was also detected. The appearance of high molecular weight lysis zones further suggested the formation of plasminogen activator-inhibitor complexes. Morphological analysis indicated that the increases in oocyte tPA activity were correlated with the extent of cumulus cell expansion and dispersion. In denuded oocytes, tPA activity also progressively increased during the periovulatory period to a maximum at 24 h after hCG treatment. In contrast, neither uPA activity nor activator-inhibitor complex was detected. Secretion of the proteases was measured in the conditioned media of cumulus-oocyte complexes cultured for 24 h in vitro. Substantial increases in tPA release were found in complexes obtained at 8 and 12 h after hCG injection, with lower secretion from complexes obtained at 24 h after hCG treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子（PAI—1）的...

Rat ovarian cells produce not only plasminogen activator (tPA) but also plasminogen activator inhibitor type 1 (PAI-1), and their coordinated geneexpression induced by gonadotropins are thought to be responsible for follicular rupture. In this study, it was demonstrated that (1) theca-interstitial compartment synthesizes the majority of PAI-1 activity in the ovary before ovulation, the follicular wall may therefore serve as a specific barrier to prevent the secretion of PA into the extrafollicular compartment; (2) Granulosa cells contribute only small amount of ovarian PAI-1 activity, but synthesize most of tissue-type plasminogen activator activity involved in the process leading to ovulation: (3) Since only matured cumulus-oocyte complexes secrete high level of tPA and PAI-1, both tPA and PAI-1 activity in the conditioned medium may be used as reliable markers for evaluating oocyte quality for in vitro fertilization.     

Vasoactive intestinal peptide stimulates plasminogen activator activity by cultured rat granulosa cells and cumulus-oocyte complexes

Vasoactive Intestinal Peptide (VIP), originally considered to be a gut hormone, has recently been found to increase estrogen and progesterone production by ovarian granulosa and luteal cells. Because several studies indicate that granulosa cells and oocytes are capable of producing plasminogen activators, we have studied the effects of VIP on plasminogen activator activity in cultured granulosa cells and cumulus-oocyte complexes collected from the ovaries of hypophysectomized, estrogen-treated immature rats. Using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by a fibrin overlay technique to assess plasminogen activator activity, we observed that treatment with VIP stimulated the secretion of tissue-type plasminogen activator (tPA), but not urinary-type plasminogen activator (uPA), in a dose-dependent manner by cultured granulosa cells as well as by cumulus-oocyte complexes, but not by denuded oocytes. However, preparation of cumulus-free oocytes from cumulus-oocyte complexes which had previously been treated with VIP indicated substantial increases in tPA activity within the oocyte. The actions of VIP on tPA activity in granulosa cells were specific, because other closely related peptides (PHM-27 and glucagon) were ineffective. These effects of VIP, in addition to the previously observed effects on steroidogenesis, suggest that VIP may be an important regulator of ovarian function.     

Elevation of plasminogen activators in cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis

A hallmark of parasitic meningitis is the infiltration of eosinophils into the subarachnoid space. Infection with Angiostrongylus cantonensis in mice induced proteinase activity in parallel with the pathological changes of eosinophilic meningitis. Zymogram analysis demonstrated that 70 and 55 kDa proteinases from cerebrospinal fluid (CSF) were active against the casein/plasminogen substrate. The proteinase activities were clearly inhibited by phenylmethanesulphonyl fluoride but not by ethylenediamine tetraacetic acid, 1,10-phenanthroline or leupeptin. Western blotting confirmed these enzymes to be tissue-type plasminogen activator and urokinase-type plasminogen activator, respectively. High activities of tissue-type plasminogen activator and urokinase-type plasminogen activator were detected in the CSF of mice with eosinophilic meningitis, and correlated positively with CSF eosinophil numbers and total protein, respectively. Immunohistochemistry demonstrated that tissue-type plasminogen activator and urokinase-type plasminogen activator localised in the endothelial cells of blood vessels, in blood clots and in infiltrated leukocytes. These results suggest that tissue-type plasminogen activator and urokinase-type plasminogen activator may be play a role in the pathogenesis of eosinophilic meningitis of angiostrongyliasis.     

Ovarian plasminogen activator: relationship to ovulation and hormonal regulation.

A technique is described for detecting fibrinolytic activity of single cells in culture. This method was applied to the analysis of rat ovarian granulosa cells. Cells obtained from follicules shortly before ovulation show high levels of fibrinolytic activity. This activity is plasminogen-dependent, indicating that it is due to plasminogen activator. The appearance of this activity is correlated with ovulation by temporal and functional criteria, and can be demonstrated both in immature animals primed with hormones and in mature cycling animals. Granulosa cell cultures can be stimulated to release plasminogen activator by exposure in vitro either to luteinizing hormone or to dibutyryl cyclic AMP.     

Plasminogen activator in early embryogenesis: Enzyme production by trophoblast and parietal endoderm

We have surveyed the early stages in the development and differentiation of cultured mouse embryos for plasminogen activator production. This enzyme is first detectable by the sixth equivalent gestation day. Thereafter, cultured blastocysts produce plasminogen activator with a biphasic time course: in the first phase, enzyme secretion rises to a maximum at about the eighth day and then decreases; a second phase, during which more enzyme accumulates, begins somewhat later and continues to at least the fifteenth day.By fractionating the blastocyst into its constituent cell types, we have identified the trophoblast as the cells responsible for the first phase of enzyme synthesis. The pattern of enzyme production by the trophoblast is closely correlated with the invasive period of these cells in vivo and implies that plasminogen activator is involved in embryo implantation. The second phase of plasminogen activator production is due to parietal endoderm, which initiates enzyme synthesis upon differentiation from the inner cell mass. The properties of the parietal endoderm suggest that plasminogen activator may participate in the migration of these cells and/or in the metabolism of Reichert's membrane which accompanies embryo growth.These results are consistent with the concept, developed from work on other cell types, that plasminogen activator may represent a generalized mechanism for tissue remodeling and cell migration.     

大鼠卵巢细胞分泌纤溶酶原激活因子抑制因子(PAI-1)的激素调节

哺乳动物卵巢合成两类纤溶酶原激活因子(PA),即组织型PA(tPA)和尿激酶型PA(uPA)。大鼠卵巢除主要合成tPA外,还分泌一种纤溶酶激活因子的抑制因子(PAI-1)。在促性腺激素作用下,卵巢tPA和PAI-1两种基因的协调表达是导致滤泡破裂的原因。本实验进一步证实,卵巢中的PAI-1主要由膜-间质细胞分泌,可能作为一种屏障限制颗粒细胞tPA分泌到滤泡外间质。当排卵来临时,两种细胞所分泌的tPA和PAI-1相互作用后仍发现有大量tPA活性。这可能是引起排卵的主要原因。因为成熟的卵丘细胞除分泌高量tPA外,还分泌大量PAI-1,在人工授精中两者有可能作为鉴定卵子优劣的可靠指标。     

Estradiol-17 beta and androgen secretion by isolated porcine ovarian follicular cells in vitro

The cellular sources and gonadotropic regulation of porcine ovarian estrogen and androgen were assessed by culturing isolated granulosa cells and thecal cells from medium size follicles (4-6 mm diameter) separately for 24 h in a chemically defined medium containing gonadotropins and (or) testosterone. At the end of the culture period, estradiol-17 beta (estradiol) and androgens in the media were determined by radioimmunoassays. Production of estradiol by granulosa cells without an exogenous aromatizable androgen was low in the absence or presence of a highly purified preparation of either follicle-stimulating hormone (FSH. 0.25 microgram/mL) or luteinizing hormone (LH. 1 microgram/mL). Addition of testosterone or androstenedione (0.5 microM), but not dihydrotestosterone or pregnenolone, significantly increased estradiol secretion. Additional increases were observed when FSH, LH, prostaglandin E2, or dibutyryl cyclic 3'.5'-adenosine monophosphate was present. Production of estradiol by thecal cells was low in the presence or absence of exogenous testosterone, and was essentially unaffected by the presence of gonadotropins. Thecal cells, however, released large amounts of androstenedione and smaller amounts of testosterone and other androgens during 24-h culture and the production of these androgens was stimulated by LH but not by FSH. Androgen secretion by granulosa cells was negligible when compared with the theca and was unaffected by gonadotropins. It is concluded that the theca is the prime site for follicular androgen biosynthesis by the porcine ovarian follicle, and, upon LH stimulation, may provide androgen precursors for estradiol production by granulosa cells.     

Altered corticosterone status impairs steroidogenesis in the granulosa and thecal cells of Wistar rats

Hypo- and hyper-corticosteronisms have adverse effects on ovarian endocrine and exocrine functions. In the present study, the mechanism by which corticosterone in excess or insufficiency impairs steroidogenesis in granulosa and thecal cells was investigated in adult albino Wistar rats. In this regard, rats were administered with corticosterone-21-acetate (2 mg/100 g b.wt., s.c., twice daily) or metyrapone (11beta-hydroxylase blocker) (10 mg/100 g b.wt., s.c., twice daily) for 15 days and a group of corticosterone/metyrapone treated rats was withdrawn of treatment and maintained for another 15 days and killed during their diestrus phase. Administration of corticosterone-21-acetate while elevated the serum corticosterone levels, metyrapone diminished the same. Administration of metyrapone reduced the serum levels of LH and estradiol; corticosterone reduced the levels of FSH in addition to LH and estradiol. In vitro production of progesterone and estradiol by the granulosa and thecal cells was decreased due to altered corticosterone status. Whereas administration of corticosterone significantly reduced the activity of 3beta-hydroxysteroid dehydrogenases (3beta-HSD) in granulosa and thecal cells, it reduced the activity of 17beta-HSD only in granulosa cells. While metyrapone treatment reduced the activity of 17beta-HSD in granulosa as well as thecal cells, it reduced the activity of 3beta-HSD only in thecal cells. The findings of the present investigation clearly demonstrate that excess or insufficiency in corticosterone affects steroidogenic process in the ovary. This is achieved by decreasing the levels of gonadotropins probably by their diminished synthesis and secretion and by interfering at the signal transduction process of these gonadotropins.     

Plasminogen activators in tissue remodeling and invasion: mRNA localization in mouse ovaries and implanting embryos

To assess in vivo the postulated participation of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators in processes involving tissue remodeling and cell migration, we have studied the cellular distribution of u-PA and t-PA mRNAs during mouse oogenesis and embryo implantation. By in situ hybridizations, we detected t-PA mRNA in oocytes and u-PA mRNA in granulosa and thecal cells from preovulatory follicles. These findings are compatible with a role for plasminogen activators in oogenesis and follicular disruption. We demonstrated the presence of u-PA mRNA in the invasive and migrating trophoblast cells of 5.5- and 6.5-d-old embryos. At 7.5 days, u-PA mRNA was predominantly localized to trophoblast cells that had reached the deep layers of the uterine wall, while the peripheral trophoblast cells surrounding the presomite stage embryo were devoid of specific signal. In 8.5-d-old embryos abundant u-PA mRNA expression resumed transiently in the giant trophoblast cells at the periphery of the embryo and in the trophoblast cells of the ectoplacental cone, to become undetectable in 10.5-d-old embryos. These observations establish the in vivo expression of the u-PA gene by invading and migrating trophoblast cells in a biphasic time pattern; they are in agreement with the proposed involvement of the enzyme in the extracellular proteolysis accompanying embryo implantation.     

Developmental regulation of Sertoli cell aromatase activity and plasminogen activator production by hormones, retinoids and the testicular paracrine factor, PModS.

Testicular peritubular cells produce a paracrine factor termed PModS that has dramatic effects on Sertoli cell function in vitro. The current study was designed to examine the actions of PModS and hormones on Sertoli cell aromatase activity and plasminogen activator production at various stages of pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats (ages correspond to prepubertal, midpubertal, and late-pubertal stages of development). Aromatase activity was found to be high and hormone-responsive in prepubertal Sertoli cells and to decline and be nonresponsive to hormones in late-pubertal Sertoli cells. FSH was the only hormone found to influence aromatase activity and estrogen production. PModS alone was not found to affect aromatase activity at any of the developmental stages examined. Interestingly, PModS was found to suppress the ability of FSH to stimulate aromatase activity and estrogen production in midpubertal Sertoli cells. Results imply that PModS may promote Sertoli cell differentiation to a more adult stage of development that is less responsive to FSH in stimulating aromatase activity. In contrast to aromatase activity, plasminogen activator production was found to increase during pubertal development. Production of Sertoli cell tissue-type plasminogen activator (tPa) was stimulated by FSH at each of the developmental stages examined, whereas production of urokinase-type plasminogen activator (uPa) was influenced by FSH only in prepubertal Sertoli cells. Insulin also stimulated uPa and tPa production by prepubertal Sertoli cells, and retinol significantly suppressed uPa production and the ability of FSH to stimulate tPa production by midpubertal Sertoli cells.     

Urokinase-type and tissue-type plasminogen activators are essential for in vitro invasion of human melanoma cells

This study evaluates the contribution of two types of plasminogen activators (PAs; tissue-type PA (tPA) versus urokinase-type PA (uPA) toward the invasiveness of human melanoma cells in a novel in vitro assay. We identified two human melanoma cell lines, MelJuso and MeWo, expressing uPA or tPA as shown at mRNA, protein, and enzyme activity level. MelJuso cells produced uPA as well as plasminogen activator inhibitor-1 (PAI-1). The latter was, however, not sufficient to neutralize the cell-associated or secreted uPA activity. MeWo cells secreted tPA, but the enzyme was not found to be cell-associated. PAI-1 production by these cells was not detectable. Plasminogen activation and fibrinolytic capacity of both cell lines were reduced by anticatalytic monoclonal antibodies specific for the respective type of PA or by aprotinin. In a novel in vitro invasion assay, antibodies to PA as well as aprotinin decreased the invasiveness of both cell lines into a fibrin gel, Matrigel, or intact extracellular matrix. Our results confirm the importance of uPA-catalyzed plasminogen activation in tumor cell invasiveness. Furthermore, we provide evidence that tPA, beyond its key role in thrombolysis, can also be involved in in vitro invasion of human melanoma cells.     

Tissue plasminogen activator mRNA in murine tissues

The urokinase-type and tissue-type plasminogen activators are the two enzymes found in mammals, which specifically convert the zymogen plasminogen to plasmin. Using cDNA probes, we have assayed for the presence of the two types of plasminogen activator mRNAs in murine tissues. We demonstrate that tissue-type plasminogen activator mRNA can be detected in a wide variety of tissues. In contrast, the accumulation of urokinase-type plasminogen activator mRNA is observed in only a few of the tissues analyzed. Using an S1 nuclease assay, we demonstrate that the tPA mRNA detected contains the complete sequences encoding the non-protease finger, growth-factor and kringle domains.     

Involvement of cytosolic phospholipase A2 in the ovulatory process in gonadotropin-primed immature rats

The preovulatory LH surge induces a remarkable increase in ovarian prostaglandins (PGs) which help to mediate the ovulatory process. We investigated whether cytosolic phospholipase A2 (cPLA2) has a role in this PG production in PMSG/hCG-primed immature rats. The immunoreactive signal for cPLA2 was localized in both thecal and granulosa layers of mature follicles and became evident in response to gonadotropins. The PLA2 activity in the whole ovarian cytosol rose slightly after PMSG stimulation, persisted relatively constant until 24 h after hCG injection and thereafter increased gradually. Intra-ovarian bursal injection of arachidonyl trifluoromethyl ketone, a specific inhibitor for cPLA2 ( 1.0-3.0 mg/ovary), significantly reduced ovarian PGE2 content and the ovulation rate. These results suggest that cPLA2 exists in periovulatory follicles and functions in PG production related to the ovulation process.     

Regulation of serine protease inhibitor-E2 and plasminogen activator expression and secretion by follicle stimulating hormone and growth factors in non-luteinizing bovine granulosa cells in vitro.

During ovarian follicle growth, there is expansion of the basal lamina and changes in the follicular extracellular matrix (ECM) that are mediated in part by proteolytic enzyme cascades regulated by tissue-type plasminogen activator (tPA) and urokinase plasminogen activator (uPA). One PA inhibitor, serine protease inhibitor-E2 (SERPINE2) is expressed in granulosa but not theca cells, and expression changes with follicle development. In this study, we hypothesized that PA and SERPINE2 expression/secretion by granulosa cells are regulated by FSH and growth factors. SERPINE2 mRNA and protein levels, tPA gene expression and uPA secretion were stimulated by FSH. Insulin-like growth factor-I stimulated SERPINE2 secretion and uPA activity, and decreased secreted tPA activity and gene expression. Bone morphogenetic protein-7 increased SERPINE2 secretion and expression and tPA secretion. In contrast, fibroblast growth factor-2 inhibited tPA secretion and SERPINE2 secretion and expression. Epidermal growth factor inhibited SERPINE2 secretion and expression, but increased secreted tPA activity. Estradiol and SERPINE2 secretion were highly positively correlated, but estradiol did not alter SERPINE2 expression. These data demonstrate that SERPINE2 expression and protein secretion are regulated by FSH and growth factors in non-luteinizing bovine granulosa cells. As estradiol is a known marker of follicle health, and SERPINE2 is an anti-apoptotic factor, we propose that SERPINE2 is involved in the regulation of atresia in bovine follicles.