Accumulation Of Vitellogenin mRNA Research Articles

Induction of vitellogenin synthesis in Locusta migratoria by the juvenile hormone analog, pyriproxyfen

Several aspects of the activity of the juvenile hormone mimic, pyriproxyfen, particularly related to the induction of vitellogenin synthesis in the fat body, have been studied in Locusta migratoria. After topical application in acetone to the neck cuticle of adult locusts, pyriproxyfen is taken up rapidly, reaching roughly constant levels in hemolymph and fat body in 6 h. For induction of vitellogenin synthesis in fat body, and for accumulation of vitellogenin in hemolymph, the ED 50 in adult females is 2.3 μg, whereas adult males produce no vitellogenin with doses up to 300 μg. In fifth-instar larvae, in contrast, the ED 50 in females is about 30 μg, and males can also be induced to produce vitellogenin, but in a smaller amount which may be related to gene dosage. With pyriproxyfen, as with methoprene, there is a lag of about 24 h before vitellogenin appears in hemolymph after primary stimulation and a reduced lag time after secondary stimulation. The accumulation of vitellogenin mRNA in fat body, assayed by hybridization with a DNA probe, shows a lag of nearly 24 h, which is extended when protein synthesis is temporarily blocked with cycloheximide. The parallel responses to pyriproxyfen and to methoprene in several different tests suggest that, despite their different structures, both compounds share the same primary mode of action, which is believed to correspond to that of natural juvenile hormone.

Hormonal control of vitellogenin mRNA levels in the male and female housefly, Musca domestica

The temporal changes and hormonal control of vitellogenin gene transcript (vitellogenin mRNA) levels in the female and male housefly, Musca domestica, were investigated during the first cycle of oögenesis by Northern blot analysis using housefly vitellogenin cDNA as a probe. The vitellogenin mRNA in the ovary and fat body in female flies accumulated in a stage-dependent fashion in parallel with the change of vitellogenin concentration in the haemolymph during egg formation. No vitellogenin mRNA was detected in the fat body of male flies. The accumulation of vitellogenin mRNA in the decapitated females of autogenous strain (in which vitellogenesis had been prevented by decapitation at the pre-vitellogenic stage) was accelerated by administration of 20-hydroxyecdysone in a dose-dependent manner. The male flies of the same strain, however, were about 100 times less sensitive to 20-hydroxyecdysone than the female flies. The time at which 20-hydroxyecdysone stimulates the vitellogenin gene was slower in males than in females. Furthermore, 20-hydroxyecdysone activated the vitellogenin gene not only in the fat body of males and females but also in the ovary of females. The vitellogenin gene expression in male flies was not affected by any dose of juvenile hormone analogue (methoprene). The female flies, however, responded to juvenile hormone analogue, though its efficacy on the induction of vitellogenin gene expression was lower and the lag time for induction was longer than those observed with the 20-hydroxyecdysone-treated female flies. The response of the ovary to juvenile hormone analogue was much stronger than that of the fat body. These results clearly indicate that the accumulation of vitellogenin mRNA in the housefly takes place in a sex-, stage- and tissue-specific fashion during the normal gonotropic cycle, and that the effect of ecdysteroid is more pronounced than that of juvenile hormone on the induction of vitellogenin gene transcription.

An estrogen-dependent polysomal protein binds to the 5′ untranslated region of the chicken vitellogenin mRNA

An estrogen-dependent protein present in chicken liver polysomes binds to the 5' untranslated region of the chicken vitellogenin II mRNA. Competition binding assays with different RNAs indicate that the binding of the polysomal protein to this region is sequence specific. Of the tissues tested, this RNA binding activity is liver specific. In vivo kinetics of appearance of the binding activity following a single injection of estrogen to immature chicks are similar to the rate of accumulation of vitellogenin mRNA. The molecular weight of the polysomal protein has been estimated to be 66,000 on the basis of UV crosslinking and subsequent SDS polyacrylamide gel electrophoresis. In vitro RNA decay assays carried out with a minivitellogenin mRNA suggest that the estrogen-dependent polysomal protein may be involved in the estrogen-mediated stabilization of the chicken vitellogenin II mRNA.

Vitellogenin gene expression in primary culture of male rainbow trout hepatocytes

Using a primary culture of trout hepatocytes we compare the kinetics of accumulation of vitellogenin and its messenger RNA after estrogen administration. We found that the cells were more sensitive to estradiol than to other estrogens. The lowest effective concentration of estradiol was 10 −9 M. At 10 −6 M the androgens have no effect. Comparison of the primary and secondary stimulation with E 2 shows that the initial rate of accumulation of vitellogenin is very much higher in the secondary stimulation. Over a time course of primary stimulation we show that after estradiol withdrawal the rate of accumulation of vitellogenin mRNA in the secondary is a function of time of the first stimulation.

Transformation of NIH 3T3 cells by micro-injected H- ras p21 protein : [formula omitted]∗ and [formula omitted]†. ∗Roche Institute of Molecular Biology, Nutley, N.J. 07110, †Department of Molecular Genetics, Roche Research Center, Nutley, N.J. 07110

From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males.Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen.During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of 32PO4 into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA.In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be <0.03% of the cytoplasmic poly(A)-containing RNA. Secondary stimulation by estrogen of these animals results in an at least 30 fold faster accumulation of vitellogenin mRNA in the cytoplasm within the initial 12 hr of hormone treatment. This may explain the faster appearance of vitellogenin in the blood plasma.

Regulation by estrogen receptor of vitellogenin gene transcription in Xenopus hepatocyte cultures

We have used primary cell cultures of hepatocytes from male or female Xenopus laevis to study the mechanisms by which estrogen induces vitellogenin gene transcription and how primary exposure to estrogen renders cells more responsive to secondary stimulation. We have characterized the estrogen receptor in hormonally narve cells and in hepatocytes treated with estrogen under a variety of conditions. Under all conditions the receptor has a K d ∼- 4 × 10 −10 M . Hormonally naïve male cells contain 300 binding sites whereas female cells or male cells previously exposed to estradiol exhibit 6–7-fold higher levels. In parallel cultures, the absolute rate of vitellogenin gene transcription was determined by hybridization of newly synthesized RNA pulse-labelled with [ 3H]uridine to cloned Xenopus vitellogenin cDNA. Naïve male cells on primary stimulation with estradiol synthesized vitellogenin mRNA at an average rate of ~ 150 moles/cell/h compared to 1200 moles/cell/h for cells previously exposed to estrogen, thus bearing a close correlation with receptor number. Furthermore, we show that the kinetics of the induced up-regulation of receptor exactly parallel those of the increase in the rate of vitellogenin gene transcription upon secondary hormonal stimulation following various periods of primary exposure to estrogen. Addition of cycloheximide to cell cultures during primary estrogen treatment abolishes both receptor up-regulation and increased rate of vitellogenin gene transcription on secondary stimulation. In addition, primary treatment with the antiestrogen tamoxifen prevents both receptor up-regulation and an enhanced rate of transcription or accumulation of vitellogenin mRNA on secondary hormonal exposure. These results demonstrate that estrogen treatment of male Xenopus hepatocytes results in the rapid up-regulation of its own receptor to female levels via new receptor synthesis, and that receptor number is rate-limiting in vitellogenin gene transcription.

Transient paralysis by heat shock of hormonal regulation of gene expression.

We have investigated the effect of heat shock on primary cultures of male and female Xenopus laevis hepatocytes as a function of estrogen-induced vitellogenin gene expression. Coincident with the induction of heat-shock protein (hsp) synthesis, thermal stress abolishes the estrogen activated transcription and accumulation of vitellogenin mRNA, at the same time causing the destabilization of vitellogenin mRNA accumulated by prior treatment with the hormone. Exposure of the cells to estrogen before heat shock allows an immediate resumption of vitellogenin gene transcription on return to 26 degrees C. Heat shock applied to cells from hormonally naive male Xenopus extends the lag period preceding vitellogenin gene transcription upon return to normal temperatures. This transient and reversible paralysis of estrogen responsiveness is paralleled by reversible changes in the amount of nuclear estrogen receptor in the hepatocytes. Heat shock therefore offers a novel approach in the manipulation and analysis of the early stages of steroid hormonal regulation of gene expression.

Unequal activation by estrogen of individual Xenopus vitellogenin genes during development

Using a technique of filter hybridization under very stringent conditions to HindIII fragments of complementary DNA cloned in plasmids, we have measured the accumulation in hepatocytes of mRNA specified by each of the four vitellogenin genes (A1, A2, B1, B2) at different stages of development of Xenopus laevis. The ontogenic competence of embryonic liver to respond to the first exposure to estradiol-17β, in terms of activation of transcription of this multigene family, is acquired late in metamorphosis at around Nieuwkoop-Faber stage 58. Upon hormonal induction, the four mRNAs accumulate under non-steady-state conditions at different rates and to different extents at all developmental stages in vivo and in cultured adult hepatocytes. A1 and B1 mRNAs appear more rapidly and accumulate to levels that are five- to eightfold those specified by genes A2 and B2, with higher amounts of B1 than A1 mRNA. A threefold higher absolute rate of synthesis of A1 and B1 mRNAs in hepatocyte cultures, relative to the A2–B2 pair, suggests that hormonal regulation of differential accumulation of vitellogenin mRNA occurs at the transcriptional level. At the early developmental stages (up to stage 61) of acquired competence, there appears to be no fixed pattern of expression, but a pattern of unequal activation of individual genes of the Xenopus vitellogenin multigene family is established thereafter and then retained at all developmental stages of tadpoles, froglets, and in both male and female adults.

Rapid estrogen metabolism and vitellogenin gene expression in xenopus hepatocyte cultures

Male hepatocytes metabolized estradiol-17β, 17α-ethinylestradiol and mestranol extremely rapidly ( t 1 2 = 40, 60 and 300 min , respectively), whereas these were more stable in cultures of female hepatocytes ( t 1 2 = 120, 150 and 640 min , respectively). Vitellogenin mRNA accumulated for only 12 h after a single addition of 10 p-6 M estradiol to male hepatocyte cultures; mestranol, but not 17α-ethinylestradiol or diethylstilbestrol, was more potent than the natural hormone. The level and rate of accumulation of vitellogenin mRNA were 5–15 times higher in female than in male hepatocytes, mestranol and estradiol being more potent than 17α-ethinylestradiol and diethylstilbestrol. Ovariectomy, 60 days prior to cell culture, did not alter the metabolism of estradiol or the vitellogenic response of female hepatocytes. On the other hand, a single administration of estradiol in vivo to male Xenopus caused a long-lasting shift (at least 16 weeks) to the female pattern of its metabolism, although the enhanced inducibility of vitellogenin genes was partially reversed between 4 and 16 weeks after hormonal treatment. The addition of fresh estradiol every 4 h to male hepatocyte cultures to compensate for its rapid metabolism resulted in a continuous and sustained accumulation of vitellogenin mRNA at rates comparable to those attained in vivo. Our findings explain the requirement for high levels of estrogen to activate vitellogenin genes and establish Xenopus hepatocyte cultures as a reproducible system for analysing the expression of this multigene family.

The role of estrogen receptor in Xenopus laevis vitellogenin gene expression.

Administration of estradiol 17 beta [estra-1,3,5(10)-triene-3,17-beta-diol] to male Xenopus laevis induces the massive synthesis by the liver of the egg yolk precursor phospholipoglycoprotein, vitellogenin, and its cognate mRNAs. Restimulation of male X. laevis that have been previously induced to synthesize vitellogenin mRNA but are inactive in vitellogenin mRNA synthesis at the time of restimulation with estrogen results in more rapid accumulation of vitellogenin mRNA and more efficient transcription of the vitellogenin genes than occurs following primary estrogen stimulation. The estrogen receptor system that mediates estrogen action in this organism exhibits several unusual properties. The cytoplasm of unstimulated liver cells contains high levels of a middle-affinity estrogen-specific binding protein and little if any estrogen receptor. The properties of the estrogen binding protein are consistent with a role in protecting estradiol 17 beta against metabolism, as a fraction of cytoplasmic estradiol 17 beta is not subject to rapid metabolism. In addition, similar binding activities are found in all Xenopus tissues surveyed that respond to steroid hormones. The induction of nuclear estrogen receptor is coincident with the onset of vitellogenin mRNA accumulation. However, an increased level of estrogen receptor is not responsible for the elevated rate of vitellogenin gene transcription observed following restimulation with estrogen.

The role of estrogen receptor in Xenopus laevis vitellogenin gene expression

Administration of estradiol 17 beta [estra-1,3,5(10)-triene-3,17-beta-diol] to male Xenopus laevis induces the massive synthesis by the liver of the egg yolk precursor phospholipoglycoprotein, vitellogenin, and its cognate mRNAs. Restimulation of male X. laevis that have been previously induced to synthesize vitellogenin mRNA but are inactive in vitellogenin mRNA synthesis at the time of restimulation with estrogen results in more rapid accumulation of vitellogenin mRNA and more efficient transcription of the vitellogenin genes than occurs following primary estrogen stimulation. The estrogen receptor system that mediates estrogen action in this organism exhibits several unusual properties. The cytoplasm of unstimulated liver cells contains high levels of a middle-affinity estrogen-specific binding protein and little if any estrogen receptor. The properties of the estrogen binding protein are consistent with a role in protecting estradiol 17 beta against metabolism, as a fraction of cytoplasmic estradiol 17 beta is not subject to rapid metabolism. In addition, similar binding activities are found in all Xenopus tissues surveyed that respond to steroid hormones. The induction of nuclear estrogen receptor is coincident with the onset of vitellogenin mRNA accumulation. However, an increased level of estrogen receptor is not responsible for the elevated rate of vitellogenin gene transcription observed following restimulation with estrogen.

Vitellogenin gene expression in male xenopus hepatocytes during primary and secondary stimulation with estrogen in cell cultures

Primary cultures of male Xenopus liver parenchymal cells that retained their competence to respond to estrogen were used to study the hormone-induced activation of the vitellogenin gene in vitro. The accumulation of vitellogenin mRNA in these cells was monitored by a quantitative diazotized paper disc hybridization procedure with a sensitivity of at least 6 pg of sequences complementary to the probe in total RNA samples of 10 μg. A short-term timecourse analysis showed that vitellogenin mRNA was detectable within 3 hr of exposure to estrogen during primary stimulation, and that the maximum rate of accumulation was reached at 5–6 hr. A longterm time-course analysis of the accumulation of vitellogenin mRNA showed that it is possible to obtain a primary response, a hormone withdrawal effect and an enhanced secondary response in the same batch of cells in a manner analogous to that observed in vivo. Measurement of hormone concentration dependence showed a response at 10 −9 M estradiol, which continued to increase up to at least 10 −6 M estradiol. This requirement for large doses of estradiol for maximal response can be explained by the rapid metabolism of estradiol by the cultured cells.

Induction of estrogen receptor and reversal of the nuclear/cytoplasmic receptor ratio during vitellogenin synthesis and withdrawal in Xenopus laevis.

The levels of cytoplasmic and nuclear estrogen receptor have been determined in livers of male Xenopus laevis stimulated by estradiol-17 beta to synthesize vitellogenin mRNA. Estrogen receptor levels were also determined in unstimulated liver and following long term withdrawal of estrogen. In unstimulated liver cells, which do not contain detectable vitellogenin mRNA, more than 80% of the estrogen receptor is located in the nucleus (550 high affinity estrogen binding sites/nucleus), while the cytoplasm contains only 100 high affinity estrogen binding sites/cell. Administration of estradiol-17 beta, which induces massive synthesis and accumulation of vitellogenin mRNA, induces the estrogen receptor as well. The nuclear receptor level rises to approximately 2,000 estrogen binding sites/cell, while the cytosol receptor increases to only 150 sites/cel. Liver cells of male X. laevis which have been withdrawn from estrogen for 70 days exhibit a striking change in receptor levels. The nuclear receptor returns to the level prevailing in unstimulated cells (approximately 500 sites/cell) while the cytosol receptor level rises to more than 1,200 sites/cell (equivalent to 260 fmol/g of tissue). The existence of a pool of cytosol receptor, which is rapidly available for induction of vitellogenin mRNA, may in part explain the shorter lag period and more rapid induction of vitellogenin mRNA observed during secondary estrogen stimulation of withdrawn Xenopus liver cells.

Multiple response patterns to oestrogenic stimulation in the avian liver

The response patterns of vitellogenin, very low density lipoprotein-apoprotein B (apo VLDL-B), and serum albumin were examined following oestrogen administration to roosters. Vitellogenin synthesis was not detectable in the absence of hormone treatment, but increased to a maximum within 3–6 days following oestrogen treatment. The hormone-stimulated increase in vitellogenin synthesis was paralleled by the accumulation of vitellogenin mRNA. In contrast to the complete oestrogen-dependence of vitellogenin synthesis, an appreciable level of apo VLDL-B synthesis was seen in the absence of exogenous hormone treatment. Following oestrogen treatment, the hepatic synthesis of apo VLDL-B increased to an extent similar in magnitude to the increase in vitellogenin synthesis. In the case of apo VLDL-B, oestrogen appears to modulate the expression of a gene which is expressed to a significant extent in the absence of the hormone. In contrast to the positive effects of oestrogen on vitellogenin and apo VLDL-B, the hepatic content of serum albumin mRNA was seen to decrease following hormone treatment. Comparison of the response patterns of these proteins suggests that oestrogen may exhibit multiple modes of control over the expression of different genes within the same cell.

Estradiol-induced accumulation of vitellogenin mRNA and secretion of vitellogenin in liver cultures of Xenopus

Expiants of male Xenopus liver maintained in a serum-free culture medium respond to stimulation by 2 × 10 −8 M 17β-estradiol with an increasing rate of accumulation of vitellogenin mRNA, as revealed by hybridization of cDNA to the total cytoplasmic RNA extracted from the cultures. A similar response is observed for secretion of 32PO 4-labeled vitellogenin into the culture medium. The in vitro response is improved in liver tissue of prestimulated animals, and by adaptation of liver expiants to the culture medium prior to hormone treatment, but attains only about 10% of the in vivo response. Since essential features of the in vivo response are maintained in liver expiants, organ culture appears suitable for investigating initial events of estradiol action leading to enhanced synthesis of vitellogenin.

Appearance of vitellogenin mRNA sequences and rate of vitellogenin synthesis in chicken liver following primary and secondary stimulation by 17 beta-estradiol.

1 Vitellogenin mRNA present in total liver RNA was determined by hybridization with labeled DNA complementary to vitellogenin mRNA. 2 In the liver of control chicks less than 0.0001% of total cellular RNA hybridized with the vitellogenin cDNA probe. About 6 h after a first estradiol injection (primary stimulation) a significant increase in vitellogenin mRNA was observed. The initial rate of accumulation of mRNA was approximately two molecules per minute per gene, 48 h later we measured a total of 7000 mRNA molecules per diploid genome (or 0.075% of total RNA). 3 Secondary stimulation was initiated 3 weeks later when the concentration of vitellogenin mRNA had returned to the baseline of controls. Upon the second injection of estradiol (secondary stimulation) an immediate increase in the number of vitellogenin mRNA sequences was observed. The initial rate of accumulation of mRNA was approximately six molecules per minute per gene, reaching 48 h later a total of 14000 mRNA molecules per diploid genome (or 0.128% of total cellular RNA). 4 The relative rate of vitellogenin synthesis in vivo was studied by pulse labeling and radioimmunochemical techniques. During the primary stimulation the rate of vitellogenin synthesis in vivo parallels the increase in vitellogenin mRNA concentration and 48 h later vitellogenin represented about 9% of the newly synthesized liver proteins. During the secondary stimulation the rate of vitellogenin synthesis increased first slowly during the first 6 h and then it ran parallel to the accumulation of vitellogenin mRNA. Two days later vitellogenin represented about 16% of the pulselabeled liver proteins.

Kinetics of avian vitellogenin messenger RNA induction. Comparison between primary and secondary response to estrogen.

Following either primary or secondary stimulation of cockerels with 17beta-estradiol, vitellogenin mRNA begins to accumulate in the liver after about 30 min, reaches a maximum in 3 days, and decays thereafter with a half-life of 30 h. During primary induction, accumulation of vitellogenin mRNA begins at a low rate (50 nucleotides/s/nuclear equivalent of DNA) and after 4 h, shifts to a higher rate (340 nucleotides/s/nuclear equivalent of DNA). In contrast, during secondary induction (administration of estrogen several weeks after the primary response has ceased), accumulation of vitellogenin mRNA begins at the rate of 350 nucleotides/s/nuclear equivalent of DNA and subsequently increases by about 40%. These accumulation rates result in a maximal level of vitellogenin mRNA that is approximately 1.5 times higher during secondary stimulation than that found during primary stimulation. This difference is sufficient to explain the anamnestic response to secondary hormonal stimulation that results in higher levels of circulating vitellogenin in the plasma of the rooster.

Quantitation of vitellogenin messenger RNA in the liver of male Xenopus toads during primary and secondary stimulation by estrogen.

Abstract From livers of estrogen-stimulated female Xenopus toads, large quantities of estrogen-induced, poly(A)-containing RNA could be isolated, showing the same characteristics as vitellogenin mRNA obtained from hormone-treated males. Using cDNA hybridization, vitellogenin mRNA was monitored in the cytoplasmic poly(A)-containing RNA of the liver of male toads during 13 days of primary and the initial phase of secondary stimulation with estrogen. During primary stimulation, low amounts of vitellogenin mRNA, not exceeding 0.18% of the cytoplasmic poly(A)-containing RNA, were first detected after 12 hr of hormone treatment, and vitellogenin mRNA was found to increase on the average to 34% of the cytoplasmic poly(A)-containing RNA on the seventh day of hormone treatment. After 3 days of primary stimulation, accumulation of vitellogenin mRNA leveled off, showing no significant increase in the cytoplasm up to 13 days of hormone treatment. As judged from incorporation of 32 PO 4 into blood plasma proteins of males during primary stimulation, vitellogenin was first detected after 1 day, and its synthesis was found to increase dramatically until the thirteenth day of hormone treatment. This implies that there is a coincidence between appearance and extent of synthesis of vitellogenin and the abundance of vitellogenin mRNA in the cytoplasm, but there is evidence that during later phase of primary stimulation (day 3–13), the increase in synthesis of vitellogenin cannot be attributed anymore to a significant accumulation of vitellogenin mRNA. In male Xenopus, estrogen-induced synthesis of vitellogenin is no more detectable 41 days after hormone injection, and the concentration of vitellogenin mRNA was found to be