Plasminogen activator in mouse and rat oocytes: induction during meiotic maturation

We have found that ovulated mouse and rat oocytes contain tissue-type plasminogen activator (PA). Primary oocytes isolated from ovaries did not contain the enzyme. During spontaneous meiotic maturation in vitro, tissue-type PA became detectable 5 hr after germinal vesicle breakdown. Induction of tissue-type PA activity was blocked by dibutyryl-cAMP or isobutylmethyl-xanthine as well as by cycloheximide, but not by actinomycin D or alpha-amanitin. These results suggest that tissue-type PA mRNA is present in primary oocytes, and that translation of this mRNA is triggered upon resumption of meiotic maturation. Tissue-type PA catalyzed proteolysis around live secondary oocytes and fertilized eggs, indicating secretion of the enzyme. Unlike secondary oocytes, fertilized eggs denuded of their zona pellucida no longer contained the enzyme, suggesting that tissue-type PA production stops at or around fertilization, and that the bulk of the enzyme is secreted at this time

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

Sites of synthesis of urokinase and tissue-type plasminogen activators in the murine kidney

Kidneys have long been recognized as a major source of plasminogen activators (PAs). However, neither the sites of synthesis of the enzymes nor their role in renal function have been elucidated. By the combined use of zymographies on tissue sections and in situ hybridizations, we have explored the cellular distribution of urokinase-type (u-PA) and tissue-type (t-PA) plasminogen activators and of their mRNAs in developing and adult mouse kidneys. In 17.5-d old embryos, renal tubules synthesize u-PA, while S-shaped bodies produce t-PA. In the adult kidney, u-PA is synthesized and released in urine by the epithelial cells lining the straight parts of both proximal and distal tubules. In contrast, t-PA is produced by glomerular cells and by epithelial cells lining the distal part of collecting ducts. The precise segmental distribution of PAs suggests that both enzymes may be implicated in the maintenance of tubular patency, by catalyzing extracellular proteolysis to prevent or circumvent protein precipitation

Involvement of the plasminogen activator/plasmin proteolytic cascade in fertilization

At the time of fertilization both murine gametes express plasminogen-dependent proteolytic activity: unfertilized eggs secrete tissue-type plasminogen activator and ejaculated spermatozoa have urokinase-type plasminogen activator bound to their surface. We now report that plasminogen is present in the fertilization environment and that both spermatozoa and eggs are able to specifically bind plasminogen. Furthermore, in vitro fertilization of mouse eggs is inhibited by antibodies which inhibit the catalytic activity of plasmin. Finally, with two different in vitro fertilization protocols, the addition of plasminogen to the fertilization medium increases the yield of fertilized eggs. These results provide evidence for a role of the plasminogen activator/plasmin proteolytic cascade in mammalian fertilization

Plasminogen activators in the mouse mammary gland. Decreased expression during lactation

The enzyme content and mRNA level for both urokinase-type and tissue-type plasminogen activators have been explored during the life cycle of the adult mouse mammary gland. Both enzymes were detected, and urokinase-type plasminogen activator was the predominant form. A marked decrease in enzyme content occurred in late gestation and was maintained throughout lactation; upon weaning, the enzyme content returned to the levels found in virgin mice. These effects were entirely accounted for by changes in the respective mRNA concentrations, which were determined with respect to both total tissue RNA and poly(A+) mRNA. Thus, plasminogen activator-catalyzed proteolysis may occur at high levels throughout the life cycle of the mouse mammary gland, except during lactation

Plasminogen activator and mouse spermatozoa: urokinase synthesis in the male genital tract and binding of the enzyme to the sperm cell surface

Abstract. When ejaculated mouse spermatozoa were embedded in a plasminogen-containing insoluble protein substrate, a zone of proteolysis developed progressively, centered around the sperm head region. Lysis did not occur in absence of plasminogen or in presence of antibodies against the urokinase-type plasminogen activator (u-PA). Zymographic and immunological analyses confirmed the presence of u-PA in extracts of ejaculated mouse spermatozoa. In contrast, the u-PA activity of sperm cells obtained from testis or from vas deferens was low, although these cells were able to bind added murine u-PA. The sites of u-PA synthesis were identified by measuring u-PA activity and u-PA mRNA content in protein extracts and in total RNA preparations of various portions of th

Meiotic maturation of mouse oocytes triggers the translation and polyadenylation of dormant tissue-type plasminogen activator mRNA

The serine protease tissue-type plasminogen activator (t-PA) is synthesized by murine oocytes undergoing meiotic maturation, but not by arrested primary oocytes. Dormant, stable t-PA mRNA accumulates during oocyte growth, so that fully grown, arrested primary oocytes contain in their cytoplasm approximately 10,000 copies of this molecule. Translation of t-PA mRNA is triggered upon resumption of meiosis and is accompanied by a progressive and concerted increase in its size. This structural change can be accounted for by increased polyadenylation at the 3' end of the molecule. Following its translation, t-PA mRNA is degraded; it is no longer detectable in fertilized eggs. The identification of a dormant mRNA in murine oocytes and the demonstration that its translational activation is accompanied by elongation of its poly(A) tail may provide insights into the control of gene expression during meiotic maturation and early mammalian development

In vivo antisense oligodeoxynucleotide mapping reveals masked regulatory elements in an mRNA dormant in mouse oocytes

In mouse oocytes, tissue-type plasminogen activator (tPA) mRNA is under translational control. The newly transcribed mRNA undergoes deadenylation and translational silencing in growing oocytes, while readenylation and translation occur during meiotic maturation. To localize regulatory elements controlling tPA mRNA expression, we identified regions of the endogenous transcript protected from hybridization with injected antisense oligodeoxynucleotides. Most of the targeted sequences in either the 5' untranslated region (5'UTR), coding region, or 3'UTR were accessible to hybridization, as revealed by inhibition of tPA synthesis and by RNase protection. Two protected regions were identified in the 3'UTR of tPA mRNA in primary oocytes: the adenylation control element (ACE) and the AAUAAA polyadenylation signal. These sequences were previously shown to be involved in the translational control of injected reporter transcripts. During the first hour of meiotic maturation, part of the ACE and the AAUAAA hexanucleotide became accessible to hybridization, suggesting a partial unmasking of the 3'UTR of this mRNA before it becomes translationally competent. Our results demonstrate that in vivo antisense oligodeoxynucleotide mapping can reveal the dynamics of regulatory features of a native mRNA in the context of the intact cell. They suggest that specific regions in the 3'UTR of tPA mRNA function as cis-acting masking determinants involved in the silencing of tPA mRNA in primary oocytes