Molecular cloning and expression of prostaglandin F2α receptor isoforms during ovulation in the ovarian follicles of Xenopus laevis.

Prostaglandins F2α levels increase during ovulatory period in Xenopus laevis in response to stimulation by gonadotropins and progesterone. PGF2α exerts its effects on ovulation through interaction with its receptor (FP) in ovaries. Little is known about the characteristics of the FP receptor and its regulation during the ovulatory period in non-mammalian species. In the present study, two isoforms of prostaglandin F receptor (FP A and B) cDNAs were isolated from Xenopus laevis ovarian tissues using reverse transcription-polymerase chain reaction (RT-PCR) followed by rapid amplification of cDNA ends (RACE). The cDNAs of FP A and FP B were sequenced. In Xenopus laevis ovary, FP A and B mRNA levels were up-regulated during gonadotropin- and progresterone-induced ovulation in vitro. The mRNA level of FP B was higher than that of FP A. Moreover, FP A and FP B mRNA levels were measured in various tissues including eye, liver, lungs, heart, muscle, ovary, and skin. Overall, FP B mRNA level was approximately 10- to 100-fold higher than that of FP A, except in the muscle and skin where FP A mRNA level was comparable to that of FP B. The results suggest that in Xenopus ovarian follicles FP receptors play an important role during gonadotropin- and progesterone-induced ovulation.

Effects of RU486 on cyclooxygenase-2 gene expression, prostaglandin F2alpha synthesis and ovulation in Xenopus laevis.

RU486 is a synthetic analog of progesterone and functions as a progesterone receptor antagonist. It binds to the progesterone receptor to prevent progesterone from occupying its receptor in many cellular systems. Early studies from our laboratory have shown that in Xenopus laevis ovarian follicles progesterone stimulates the expression of cyclooxygenase-2 (COX-2) gene which leads to a rapid increase in the production of prostaglandin F2alpha (PGF2alpha) and subsequent ovulation. In this study, we examined the effect of RU486 on the synthesis of COX-2 mRNA, production of PGF2alpha and ovulation in X. laevis. Ovarian tissue fragments were primed with human chorionic gonadotropin (hCG) and then incubated with progesterone (P4) alone or in the presence of varying concentrations of RU486 over a period of 12h. After the incubation ovulated oocytes were counted, COX-2 expression and synthesis of PGF2alpha were measured. Results demonstrated that RU486 attenuated the expression of COX-2 gene, reduced the synthesis of PGF2alpha, and inhibited ovulation in a dose-dependent manner. This finding suggests that progesterone receptor is an important regulator in the progesterone-cyclooxygenase-prostaglandin-mediated ovulation in amphibians.

Lateral gene transfer and parallel evolution in the history of glutathione biosynthesis genes.

BACKGROUND: Glutathione is found primarily in eukaryotes and in Gram-negative bacteria. It has been proposed that eukaryotes acquired the genes for glutathione biosynthesis from the alpha-proteobacterial progenitor of mitochondria. To evaluate this, we have used bioinformatics to analyze sequences of the biosynthetic enzymes gamma-glutamylcysteine ligase and glutathione synthetase. RESULTS: Gamma-glutamylcysteine ligase sequences fall into three groups: sequences primarily from gamma-proteobacteria; sequences from non-plant eukaryotes; and sequences primarily from alpha-proteobacteria and plants. Although pairwise sequence identities between groups are insignificant, conserved sequence motifs are found, suggesting that the proteins are distantly related. The data suggest numerous examples of lateral gene transfer, including a transfer from an alpha-proteobacterium to a plant. Glutathione synthetase sequences fall into two distinct groups: bacterial and eukaryotic. Proteins in both groups have a common structural fold, but the sequences are so divergent that it is uncertain whether these proteins are homologous or arose by convergent evolution. CONCLUSIONS: The evolutionary history of the glutathione biosynthesis genes is more complex than anticipated. Our analysis suggests that the two genes in the pathway were acquired independently. The gene for gamma-glutamylcysteine ligase most probably arose in cyanobacteria and was transferred to other bacteria, eukaryotes and at least one archaeon, although other scenarios cannot be ruled out. Because of high divergence in the sequences, the data neither support nor refute the hypothesis that the eukaryotic gene comes from a mitochondrial progenitor. After acquiring gamma-glutamylcysteine ligase, eukaryotes and most bacteria apparently recruited a protein with the ATP-grasp superfamily structural fold to catalyze synthesis of glutathione from gamma-glutamylcysteine and glycine. The eukaryotic glutathione synthetase did not evolve directly from the bacterial glutathione synthetase.