Regulation of the Yp subunit of glutathione S-transferase P in rat embryos and yolk sacs during organogenesis.
Manipulation of the glutathione status of an embryo during organogenesis leads to abnormal development, as well as increasing the susceptibility of the embryo to insult by either xenobiotic or endogenous electrophiles. The glutathione S-transferases are a family of drug-metabolizing enzymes that catalyze the conjugation of reactive chemicals with glutathione, playing an important role in protecting cells against attack. The purpose of this study was to investigate the presence and regulation of one glutathione S-transferase, glutathione S-transferase P, a homodimer of the Yp subunit, in the conceptus during organogenesis. Northern blot analysis of the RNA isolated from rat embryos and their yolk sacs on days 10, 11 and 12 of gestation revealed a single Yp transcript. Steady-state concentrations of the Yp mRNA in embryos did not change with either gestational age or culture for 24 hr (day 11 in vitro) or 45 hr (day 12 in vitro). In contrast, concentrations of this transcript in yolk sac increased 3-fold from day 10 to 12 of gestation and a further 3-fold with culture (day 12 in vivo compared with in vitro). Transcription of the rat Yp subunit gene in cell lines is induced by treatment with phorbol esters. However, the addition of 12-O-tetradecanoylphorbol-13-acetate (TPA, 50 or 100 nM) to embryos in culture had no effect on the steady-state concentrations of the Yp transcript. Thus, the glutathione S-transferase Yp message is subject to tissue- and development-specific regulation in the conceptus during organogenesis. Moreover, culture of the embryos resulted in a further up-regulation of the steady-state concentrations of the Yp transcript in yolk sac. Western blot analysis demonstrated that a single immunoreactive Yp subunit band of 26 kDa was found in both embryos and yolk sacs. Neither age nor culture appeared to affect the concentrations of immunoreactive Yp subunit in the yolk sac. Thus, glutathione S-transferase Yp mRNA is translated in the conceptus during organogenesis. The apparent differences between the relative amounts of the message and immunoreactive protein in yolk sac suggest that this subunit may be subject to post-transcriptional as well as transcriptional regulation in this tissue. Immunohistochemical analysis of embryos cultured for 45 hr (day 12 in vitro) revealed that the Yp reaction product was localized over the hepatic primordia, mesonephric ducts, otocyst, yolk sac and ectoplacental cone.(ABSTRACT TRUNCATED AT 400 WORDS)
Hales
B F
BF
Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada.
Huang
C
C
D000818:Animals; D004622:Embryo, Mammalian / Q000187:drug effects / Q000201:enzymology*; D005865:Gestational Age; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007150:Immunohistochemistry; D009924:Organ Culture Techniques; D010703:Phorbol Esters / Q000494:pharmacology; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015017:Yolk Sac / Q000187:drug effects / Q000201:enzymology*
D010703:Phorbol Esters; D012333:RNA, Messenger; D005982:Glutathione Transferase