Regulation of hepatic progesterone and estrogen receptors in the female turtle, Chrysemys picta: relationship to vitellogenesis.

Previous studies using the fresh water turtle Chrysemys picta have demonstrated the differential expression of the two progesterone receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with ER mRNA levels and hepatic vitellogenesis. During the inter-vitellogenic periods the ratio of PRB:PRA favors PRA, suggesting that the PRB:PRA ratio may be important in the regulation of vitellogenesis. Based on these and other studies we hypothesize that progesterone may have differential effects on the estrogen (E)-induced vitellogenin response (inhibitory or stimulatory), depending on the PRB:PRA ratio. In this study, we determined the expression pattern and the hormonal regulation of progesterone receptor (PR) isoforms in the liver, during the estrogen-induced vitellogenin response in female C. picta. Northern blot analysis was used to evaluate the changes in vitellogenin mRNA, estrogen receptor (ER) mRNA, and PR mRNA expression; Western blot to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vitellogenin. The expression of PR isoforms in the liver of female turtles at the mRNA and protein levels was differentially regulated by estradiol and progesterone. Estradiol treatment enhanced the transcription of PR mRNA isoforms and possibly translation and/or increased stability of PRB protein in the female turtle liver. In contrast, this hormone decreased PRA protein levels. Progesterone alone down-regulated progesterone receptor isoform A (PRA) and progesterone receptor isoform B (PRB) proteins equally, but did not affect PR mRNA levels. Estradiol markedly increased hepatic ER mRNA, vtg mRNA, and vtg in plasma, but this effect was not impacted significantly by progesterone. The results presented here indicate that in female turtles hepatic PRB:PRA ratios are markedly changed by estradiol treatment due primarily to a decrease in PRA. The change in the PRB:PRA ratio after hormonal treatment confirms that progesterone and estrogen exposure will be a determinant in the regulation of vitellogenesis, and, in turn, that the regulation of vitellogenesis will be determined by the ratio of PR isoforms and the physiological levels of steroid hormones.

Effect of gonadal steroids on progesterone receptor, estrogen receptor, and vitellogenin expression in male turtles (Chrysemys picta).

Hepatic vitellogenin (vtg) is a yolk precursor protein sequestered in follicular oocytes as nutrient supply for developing embryos in nonmammalian vertebrates. In prior research studies we have demonstrated that both progesterone (P) and testosterone (T) inhibit estrogen (E)-induced vitellogenesis in the male fresh water turtle (Chrysemys picta), and have suggested that these hormones may be involved in multihormonal regulation of vitellogenesis in the female turtle. However, the modes of action of progesterone and testosterone on estrogen-induced vitellogenesis are not known. We have proposed that progesterone inhibits vitellogenesis by modulation of progesterone receptor A (PRA) or B (PRB) isoforms and/or estrogen receptor (ER) gene transcription. In this study, we compare the vitellogenic responses of reproductively inactive male turtles to estradiol 17beta in the presence of exogenous testosterone or progesterone. Northern blot analysis was used to monitor the changes in vtg mRNA, ER mRNA, and PR mRNA expression; Western blotting to determine changes in PR isoform expression and a homologous ELISA for measurement of plasma vtg. Progesterone and testosterone reduced estrogen-induced vtg mRNA expression, but plasma vtg was not significantly reduced by these steroids. PRA and PRB were transcribed even though ER mRNA could not be detected, suggesting constitutive PR expression. However, in the presence of estradiol 17beta, both PR isoforms and mRNA transcripts were increased as a correlate of ER mRNA transcription, suggesting both transcriptional and translational effects; these effects were inhibited by testosterone and progesterone treatments. Since ER mRNA was sharply reduced by both testosterone and progesterone, and estradiol 17 beta increased PR mRNA transcription and translation, it is likely that the action of progesterone in reducing vtg mRNA is indirect via down regulation of ER mRNA, thus ER. This study provides further information on the role of progesterone and testosterone in the regulation of hepatic vitellogenesis, suggesting regulation of vitellogenesis mainly via modulation of hepatic ER mRNA.

Seasonal changes in hepatic progesterone receptor mRNA, estrogen receptor mRNA, and vitellogenin mRNA in the painted turtle, Chrysemys picta.

Previous studies using the fresh water turtle Chrysemys picta have demonstrated that progesterone (P) inhibits estradiol (E)-induced vitellogenin (vtg) secretion in this species. Further, there is evidence for the differential expression of the two P receptor isoforms (PRA and PRB) in the liver during the turtle seasonal cycle, correlating with hepatic vitellogenesis. In this study we report changes in the hepatic PR mPNA, ER mRNA, and vitellogenin (vtg) mRNA transcripts during the reproductive cycle of the turtle. Fragments of the turtle hepatic PR and ER cDNAs were cloned and sequenced and a previously cloned turtle vtg cDNA were used as probes in Northern blotting. No 3.7-kb PR mRNA, corresponding to the smaller PR transcript, PRA of other species was found, although, a smaller 1.8-kb transcript (putative PRC mRNA) was present. These observations suggest that the turtle as in the chicken and human, the 4.5-kb PR mRNA transcript encodes both PRA and PRB proteins. Only the larger PR mRNA transcript (4.5-kb), was found to vary significantly during the annual cycle, being highest when vitellogenesis was inhibited in winter and summer. Vtg mRNA could not be detected during the summer or winter, was highest during vitellogenesis in the spring, and reappeared during the fall period of vitellogenesis and ovarian recrudescence. ER mRNA followed a similar pattern, being highest during spring and early fall, when vtg synthesis is high. The data suggest that P/PR, as well as E/ER, may be involved in the seasonal regulation of hepatic vitellogenesis in this species.

Progesterone and progesterone receptors in reptiles.

The role of progesterone (P) has been most extensively studied in the female reproductive tissues (ovary, reproductive tract, mammary gland) and in the brain, in which it is an important regulator and modulator in conjunction with estradiol (E). In nonmammalian vertebrate species, less work has been done on P metabolites involved in ovulation. In addition, P induces the expression of egg-white proteins, decreases myometrial contractility, and facilitates processing of eggs, formation of eggshell, and deposition of egg-white proteins. Actions of P may be synergistic with, or antagonist to, the actions of E, depending on hormone ratios, timing of exposure, and physiological state. These effects of P are mediated through progesterone receptor isoform A (PRA), a general transcription inhibitor of P target genes, and isoform B (PRB), a specific transcriptional stimulator of some reproductive tract genes. In this review, we focus on P action and PR expression in the liver and reproductive tissues of several reptilian species, especially Chelonia, since most of the data obtained are from this group. We also present novel data showing the expression of PR in nonreproductive tissues (such as kidney, spleen, gastrointestinal tract) in the painted turtle, Chrysemys picta.