Prolactin regulation of tissue type plasminogen activator and plasminogen activator inhibitor type-I gene expression in eCG-primed rat granulosa cells in culture.
The present study was designed to investigate the effect of prolactin (PRL) on plasminogen activator inhibitor-I (PAI-I) and tissue type plasminogen activator (tPA) gene expression in eCG-primed granulosa cells in vitro. At 46 h after the hormone treatment, ovaries were removed, and granulosa cells were prepared for culture. Cells were incubated for various times in serum-free medium in the presence or absence of LH and PRL alone or in combination. tPA and PAI-I activities in the media were assayed by fibrin overlay and reverse fibrin autograph, respectively. Cytoplasmic RNA from granulosa cells was prepared using the NP-40 method and was assayed for PAI-I and tPA mRNA levels. We demonstrated the following. 1) PRL increased PAI-I mRNA production in cultured granulosa cells. Inclusion of LH with PRL had a synergistic effect on increasing PAI-I mRNA levels. After 48-h culture, 3-fold increases in PAI-I mRNA levels were seen with LH in combination with PRL as compared with PRL alone. The synergistic increase in PAI-I mRNA levels occurred in a dose- and time-dependent manner. 2) The increase in PAI-I mRNA synthesis by PRL alone, or by PRL in combination with LH, was well correlated with the changes in PAI-I activity and antigen levels in the conditioned media. 3) PRL in the culture also dramatically decreased LH-induced tPA mRNA and activity in a dose- and time-dependent fashion. The decrease in the tPA activity by PRL was also correlated with an increase in the amount of PA-PAI-I complexes in the cell-conditioned media. 4) In situ hybridization of tPA and PAI-I mRNAs in the cultured granulosa cells also showed that PRL was capable of enhancing PAI-I mRNA while diminishing tPA mRNA production induced by LH. This suggests that the dose- and time-dependent decrease in the gonadotropin-induced tPA activity in the culture by the presence of PRL may be due to decreasing tPA mRNA synthesis on one hand and to neutralization of the tPA activity by the increased PAI-I activity on the other.
State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100080, China. liuyx@panda.ioz.ac.cn
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Liu
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Peng
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Liu
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Chen
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D000818:Animals; D001345:Autoradiography; D015153:Blotting, Western; D002478:Cells, Cultured; D015342:DNA Probes; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects*; D006064:Gonadotropins, Equine / Q000494:pharmacology*; D006107:Granulosa Cells / Q000378:metabolism*; D017403:In Situ Hybridization; D007986:Luteinizing Hormone / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics*; D011388:Prolactin / Q000502:physiology*; D015347:RNA Probes; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics*
D015342:DNA Probes; D006064:Gonadotropins, Equine; D017395:Plasminogen Activator Inhibitor 1; D015347:RNA Probes; D012333:RNA, Messenger; D011388:Prolactin; D007986:Luteinizing Hormone; D010959:Tissue Plasminogen Activator