Quantitative trait loci for estrogen-dependent pituitary tumor growth in the rat

Growth control is of fundamental importance to biology in general and of critical importance to cancer research in particular. Tumors develop when control of the normal growth process is lost. The rat pituitary is a model system for control of estrogen-dependent growth. Chronic estrogen treatment induces uncontrolled growth in the pituitaries of Fischer 344 (F344) rats, but not of Brown Norway (BN) rats. We have identified five quantitative trait loci (QTL) for estrogen-dependent pituitary mass (Edpm) in an F₂ intercross of F344 and BN. These QTL reside on rat Chromosomes (Chrs) 2, 3, 5, and 9 and explain a total of 55% of the genetic variance in the F₂. We have also detected suggestive evidence for a QTL on rat Chr 14. For Edpm2-1, Edpm2-2, Edpm3, and Edpm5, the F344 allele corresponds with increased pituitary mass, as expected. Surprisingly, for Edpm9 and the suggested QTL on Chr 14, the BN allele corresponds with increased pituitary mass. We also find evidence for interaction (epistasis) between Edpm3 and Edpm9 and between Edpm5 and the suggested QTL on Chr 14. Received: 3 March 1997 / Accepted: 1 July 1997     

Angiogenesis and capillary maturation phenotypes associated with the Edpm3 locus on rat chromosome 3

The quantitative trait locus (QTL) Edpm3 is one of a group of additively acting QTL \responsible for the difference in estrogen-induced pituitary tumor growth between the tumor-susceptible F344 and tumor-resistant BN rat strains. The F344.BN-Edpm3^BN rat strain was produced by moving the segment of rat Chr 3 between D3Mgh7 and D3Mgh13, which contains the Edpm3 QTL, from the BN strain into the F344 genetic background. In a previous study, we used this congenic line to find that the BN allele of the Edpm3 QTL reduces tissue mass and S-phase fraction in the estrogen-induced rat pituitary tumor. We now report on the use of this congenic line to investigate the linkage of Edpm3 to tumor angiogenesis. Contrary to expectation, the F344.BN-Edpm3^BN strain has significantly greater angiogenic activity than does F344 in both treated and untreated rats. Microvessel count (MVC), perivascular space, and number of nonattached pericytes/pericapillary fibroblasts are all elevated in the pituitary by chronic estrogen treatment and their values are significantly greater in F344.BN-Edpm3^BN than F344. Thus, although there is greater angiogenic activity in the pituitary of estrogen-treated F344.BN-Edpm3^BN rats, there is a deficiency in capillary maturation compared with F344.     

Different functions of QTL for estrogen-dependent tumor growth of the rat pituitary

Estrogen treatment to rats of the Fischer 344 (F344) strain induces growth of pituitary tumors that exhibit accelerated cell proliferation, breakdown of basement membrane, and formation of hemorrhagic lakes. Estrogen-dependent pituitary growth is due to variation in a group of quantitative trait loci (QTL), called Edpm for estrogen-dependent pituitary mass, that we previously identified in an F₂ intercross of F344 and the tumor-resistant Brown Norway strain. We previously identified 5 QTL, and microsatellite markers developed since our earlier work have allowed us to scan new chromosomal regions, resulting in two new QTL for estrogen-dependent pituitary mass: Edpm9-2 and a possible QTL on the X Chromosome (Chr). Here we report evidence that these QTL differ from each other in how they affect growth. To examine the effect of the Edpm QTL on biochemical components of tumor growth, we tested their effects in 138 progeny of a backcross to the F344 strain which were given a 10-week chronic estrogen treatment. Hemoglobin/DNA ratio (a measure of blood volume relative to cell number) and total pituitary DNA (a measure of cell number) correlated only weakly, and very large pituitaries were observed which had a low hemoglobin/DNA ratio resembling a normal gland. Through QTL mapping, we found that Edpm2-1, Edpm3, Edpm5, and Edpm9-2 all had significant effects on pituitary mass, but Edpm2-1 and Edpm9-2 primarily affected DNA content, Edpm5 primarily affected hemoglobin/DNA ratio, and Edpm3 affected all traits equally. Received: 16 March 2000 / Accepted: 17 May 2000     

Effects of the xenoestrogen bisphenol A on expression of vascular endothelial growth factor (VEGF) in the rat

Bisphenol-A (BPA) is used to produce polymers for production of polycarbonate and epoxy resins that are used in food containers and dental appliances. BPA binds to estrogen receptors and induces estrogenic activity in a number of biological systems. We recently reported that although Fisher 344 (F344) and Sprague-Dawley (S-D) rat strains exhibit different sensitivities to BPA at the level of vaginal epithelial cell proliferation, there was no difference in immediate early proto-oncogene expression between the two animal strains. In the present study we investigated the effects of BPA on expression of another estrogen-target gene, vascular endothelial growth factor (VEGF), in the uterus, vagina, and pituitary of F344 and S-D rats. Adult rats were ovariectomized and treated with BPA by intraperitoneal injection at concentrations of 0.02 to 150 mg/kg body wt. Expression of VEGF was monitored by RNase protection assay at 2 hr after treatment. There was a significant effect of dose of BPA on the type of VEGF isoform expressed in the uterus, vagina, and pituitary. BPA induced greater (P < 0.01) levels of VEGF164 and VEGF120+188 than VEGF110 levels. The lowest BPA dose that had a significant (P< 0.05) effect on VEGF expression compared with vehicle treatment was 37.5 mg/kg body wt.; dose-response curves did not differ between strains. This is the first report that the primary response of the uterus, vagina, and pituitary to BPA includes rapid induction of VEGF expression. Due to the capacity of VEGF to engage pleiotropic signaling pathways in other cellular systems, we suggest that modulation of VEFG may play a role in establishing the response of estrogen-target organs to estrogenic xenobiotics.     

Global analysis of gene expression in the estrogen induced pituitary tumor of the F344 rat

The F344 rat rapidly forms large prolactinomas in response to chronic estrogen treatment. To identify genes expressed in the course of this estrogen induced pituitary tumor growth, we performed microarray analysis on the F344 rat pituitary after chronic estrogen treatment and on untreated controls. At a significance level set to minimize type I error, some 72 genes were found to be differentially expressed between estrogen treated and untreated. Of those genes, 70 have not been reported previously as being affected by estrogen in the F344 rat pituitary. Since many other investigators have studied the effect of estrogen on specific gene expression in rat pituitary, we also examined the mRNA expression of the 36 genes that have been previously reported as having their expression affected by estrogen in the rat pituitary. Of these, 13 were found to have their expression affected by estrogen treatment in the same direction as had been reported by others.     

Impact of genetic background and aging on mesenteric collateral growth capacity in Fischer 344, Brown Norway, and Fischer 344 x Brown Norway hybrid rats

Available studies indicate that both genetic background and aging influence collateral growth capacity, but it is not known how their combination affects collateral growth. We evaluated collateral growth induced by ileal artery ligation in Fischer 344 (F344), Brown Norway (BN), and the first generation hybrid of F344 x BN (F1) rats available for aging research from the National Institute on Aging. Collateral growth was determined by paired diameter measurements in anesthetized rats immediately and 7 days postligation. In 3-mo-old rats, significant collateral growth occurred only in BN (35% +/- 11%, P < 0.001). The endothelial cell number in arterial cross sections was also determined, since this precedes shear-mediated luminal expansion. When compared with the same animal controls, the intimal cell number was increased only in BN rats (92% +/- 21%, P < 0.001). The increase in intimal cell number and the degree of collateral luminal expansion in BN rats was not affected by age from 3 to 24 mo. Immunohistochemical studies demonstrated that intimal cell proliferation was much greater in the collaterals of BN than of F1 rats. The remarkable difference between these three strains of rats used in aging research and the lack of an age-related impairment in the BN rats are novel observations. These rat strains mimic clinical observations of interindividual variation in collateral growth capacity and the impact of age on arteriogenesis and should be useful models to investigate the molecular mechanisms responsible for such differences.     

Inhibitory effect of thalidomide on the growth, secretory function and angiogenesis of estrogen-induced prolactinoma in Fischer 344 rats

The process of angiogenesis has been found to be essential for the development of estrogen-induced pituitary prolactinoma in Fischer 344 rats. Thalidomide [(alpha-(N-phthalimido)-glutarimide] is known to be a potent immunomodulatory drug with antiangiogenic properties, but its effect on lactotroph cell secretory function and pituitary prolactinoma formation has not been described yet. The purpose of this study was to examine the effects of thalidomide on secretion of prolactin (PRL) and vascular endothelial growth factor (VEGF), cell proliferation, apoptosis and angiogenesis within the anterior pituitary gland in long-term diethylstilboestrol (DES)-treated male F344 rats in vivo and in vitro. It was found that DES sharply increased serum PRL and VEGF levels. On the other hand, simultaneous treatment of F344 rats with thalidomide for the last 15 days of the experiment attenuated the stimulatory effect of DES on PRL and VEGF secretion. It also diminished prolactin cell proliferation evaluated as the number of proliferating cell nuclear antigen (PCNA)-positive stained cell nuclei and increased the number of apoptotic bodies determined by the terminal deoxynucleotidyl-mediated dUTP nick-end labeling (TUNEL) method in sections of the DES-induced pituitary prolactinoma. The density of pituitary microvessels evaluated by microscopic counting of CD-31-positive blood vessels was also diminished by the tested drug. In addition, thalidomide (10(-4) to 10(-6) M) inhibited cell proliferation, prolactin and VEGF secretion from rat pituitary prolactinoma cells cultured in vitro. In conclusion, our results provide strong evidence for the antiprolactin and antitumor activity of thalidomide in experimentally DES-induced pituitary adenoma.     

Estrogen stimulates both prolactin and growth hormone mRNAs expression in the MtT/F4 transplantable pituitary tumor

The effects of 17 beta-estradiol (E2) on MtT/F4 pituitary tumor growth and on prolactin (PRL) and growth hormone mRNA expression were analyzed in F344 female rats. E2 (10 mg) stimulated pituitary PRL cell hyperplasia and PRL mRNA, but inhibited growth of the transplantable tumors. The expression of both PRL and growth hormone mRNA levels was increased in the MtT/F4 tumors. The effects of E2 on increasing PRL mRNA levels were more marked in the pituitary compared with the tumors. These results indicate that estrogens stimulate proliferation and PRL expression in the pituitary while inhibiting cell proliferation in the MtT/F4 tumor. E2 also stimulated both growth hormone and PRL mRNA expression in the MtT/F4 transplantable tumor.     

A combined opiate agonist and antagonist treatment reduces prolactin secreting pituitary tumor growth

Prolactin secreting pituitary adenomas (prolactinomas) is the most common pituitary tumors in humans. Animal studies have identified aggressive prolactinoma development in fetal alcohol exposed rats. We have recently identified a combination treatment of a μ opioid receptor antagonist naltrexone and a δ opioid receptor agonist D-Ala2-,N-Me-Phe4,Gly-ol Enkephalin (DPDPE) increases innate immune function. In this study, we tested whether naltrexone and DPDPE combination therapy is useful to control pituitary tumor growth. Fetal alcohol exposed and control Fischer 344 female rats at 60 days of age were ovariectomized and received an estrogen implant to induce prolactinomas. Six weeks after the estrogen implant, these animals received treatments of naltrexone and DPDPE or saline. The growth of the pituitary tumor prior to and after opioidergic agent treatments was visualized using magnetic resonance imaging (MRI). At the end of the treatment, pituitary weights, plasma prolactin and splenic levels of cytotoxic factors were determined. Both imaging data and weight data indicated that the volume and the weight of the pituitary were increased more after estrogen treatment in animals exposed to fetal alcohol than control. Naltrexone and DPDPE treatment reduced the weight and volume of the pituitary gland and plasma levels of prolactin in both fetal alcohol exposed and control-fed animals. The treatment of opioidergic agents also increased the levels of cytotoxic factors in the spleen. These data provide a novel possibility in treating pituitary tumors using a combination therapy of naltrexone and DPDPE.     

Localization of Eutr2, a locus controlling susceptibility to DES-induced uterine inflammation and pyometritis, to RNO5 using a congenic rat strain

In some rat strains chronic administration of exogenous estrogens induces pyometritis, an inflammation of the uterus associated with infection, suggesting that there is genetic variation in susceptibility to estrogen-induced inflammation and pyometritis. In this article we report that following 10 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), Fisher 344 (F344) rats exhibit modest uterine inflammation and a 0% incidence of pyometritis. By contrast, under identical experimental conditions, Brown Norway (BN) rats exhibit significant inflammation and a 100% incidence of pyometritis. Similarly, we also observed profound uterine inflammation and a 100% incidence of pyometritis in a congenic rat strain in which a segment of RNO5 from the BN strain is carried on the F344 strain. These data suggest that a locus on RNO5 controls both the magnitude of DES-induced uterine inflammation and susceptibility to DES-induced pyometritis. This locus, designated Eutr2, resides within the same segment of RNO5 as the Eutr1 locus, which confers susceptibility to E2-induced pyometritis in an F2 population generated in a cross between the BN and August × Copenhagen 9935, Irish (ACI) strains. Jyotsna Pandey, Karen A. Gould contributed equally to this work.     

Depletion and release of prolactin from rat pituitaries and pituitary tumors in vitro

Depletion of pituitary prolactin (PRL) and PRL release into culture medium were simultaneously examined over a 3.5- to 4.0-hr incubation period from anterior pituitary fragments obtained from Fischer-344 or Wistar-Furth female rats treated with estrogen for 5 days, in pituitary tumors induced by 8 weeks of diethylstilbestrol (DES) treatment in Fischer-344 rats and in MtTW15 pituitary tumors transplanted subcutaneously in Wistar-Furth rats for 4 weeks. Our objective was to determine if the event known as transformation, which we define as a loss in the tissue PRL content without a corresponding and equivalent increase in the medium PRL content, occurs in rat pituitary tumors. Our results indicated that transformation did not occur in vitro in rat anterior pituitary tumors induced in Fischer-344 rats by DES treatment but was present in pituitaries from Fischer-344 rats treated for 5 days with estrogen, which served as controls. We also observed in vitro transformation in the anterior pituitary of Wistar-Furth rats treated with estrogen for 5 days (controls) and in the pituitaries of Wistar-Furth rats inoculated with the MtTW15 tumor for 4 weeks, but not in the MtTW15 tumor itself. Although transformation was present in both Fischer-344 and Wistar-Furth rats treated acutely with estrogen the timing of the transformation was delayed 1-2 hr in the Fischer-344 rats compared with Wistar-Furth females. We concluded that transformation does not precede release of prolactin in rat pituitary tumors and that in normal pituitaries the mechanisms of transformation are induced differently between the strains of rats examined.     

Genetic bases of estrogen-induced pituitary tumorigenesis: identification of genetic loci determining estrogen-induced pituitary growth in reciprocal crosses between the ACI and Copenhagen rat strains

Estrogens stimulate proliferation and enhance survival of the prolactin (PRL)-producing lactotroph of the anterior pituitary gland and induce development of PRL-producing pituitary tumors in certain inbred rat strains but not others. The goal of this study was to elucidate the genetic bases of estrogen-induced pituitary tumorigenesis in reciprocal intercrosses between the genetically related ACI and Copenhagen (COP) rat strains. Following 12 weeks of treatment with the synthetic estrogen diethylstilbestrol (DES), pituitary mass, an accurate surrogate marker of absolute lactotroph number, was increased 10.6-fold in ACI rats and 4.5-fold in COP rats. Composite interval mapping analyses of the phenotypically defined F(2) progeny from the reciprocal crosses identified six quantitative trait loci (QTL) that determine the pituitary growth response to DES. These loci reside on chromosome 6 [Estrogen-induced pituitary tumor (Ept)1], chromosome 3 (Ept2 and Ept6), chromosome 10 (Ept9), and chromosome 1 (Ept10 and Ept13). Together, these six Ept loci and one additional suggestive locus on chromosome 4 account for an estimated 40% of the phenotypic variance exhibited by the combined F(2) population, while 34% of the phenotypic variance was estimated to result from environmental factors. These data indicate that DES-induced pituitary mass behaves as a quantitative trait and provide information that will facilitate identification of genes that determine the tumorigenic response of the pituitary gland to estrogens.     

Effect of antiestrogens and aromatase inhibitor on basal growth of the human breast cancer cell line MCF-7 in serum-free medium

Antiestrogens are efficient inhibitors of estrogen-mediated growth of human breast cancer. Besides inhibiting estradiol-stimulated growth, antiestrogens may have a direct growth-inhibitory effect on estrogen receptor (ER) positive cells and thus be more efficient than aromatase inhibitors, which will only abrogate estrogen-dependent tumor growth. To address this issue, we have used the human breast cancer cell line MCF-7/S9 as a model system which is maintained in a chemically defined medium without serum and estrogen. The addition of estradiol results in an increase in cell growth rate. Thus, the MCF-7/S9 cell line is estrogen-responsive but not estrogen-dependent. Three different types of antiestrogens, namely tamoxifen, ICI 182,780 and EM-652 were found to exert a significant and dose-dependent inhibition of basal growth of MCF-7/S9 cells. The growth-inhibitory effect of the three antiestrogens was prevented by simultaneous estradiol treatment. Antiestrogen treatment also reduced the basal pS2 mRNA expression level, thus indicating spontaneous estrogenic activity in the cells. However, treatment with the aromatase inhibitor had no effect on basal cell growth, excluding that endogenous estrogen synthesis is involved in basal growth. These data demonstrate that in addition to their estrogen antagonistic effect, antiestrogens have a direct growth-inhibitory effect which is ER-mediated. Consequently, in the subset of ER positive breast cancer patients with estrogen-independent tumor growth, antiestrogen therapy may be superior to treatment with aromatase inhibitors which only inhibit estrogen formation but do not affect cancer cell growth in the absence of estrogens.     

Genetic dissection of ``OLETF', a rat model for non-insulin-dependent diabetes mellitus

To elucidate the genetic factors underlying non-insulin-dependent diabetes mellitus (NIDDM), we performed genome-wide quantitative trait locus (QTL) analysis, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF rat is an excellent animal model of NIDDM because the features of the disease closely resemble human NIDDM. Genetic dissection with two kinds of F2 intercross progeny, from matings between the OLETF rat and non-diabetic control rats F344 or BN, allowed us to identify on Chromosome (Chr) 1 a major QTL associated with features of NIDDM that was common to both crosses. We also mapped two additional significant loci, on Chrs 7 and 14, in the (OLETF × F344)F₂ cross alone, and designated these three loci as Diabetes mellitus, OLETF type Dmo 1, Dmo2 and Dmo3 respectively. With regard to suggestive QTLs, we found loci on Chrs 10, 11, and 16 that were common to both crosses, as well as loci on Chrs 5 and 12 in the (OLETF × F344)F₂ cross and on Chrs 4 and 13 in the (OLETF × BN)F₂ cross. Our results showed that NIDDM in the OLETF rat is polygenic and demonstrated that different genetic backgrounds could affect ``fitness' for QTLs and produce different phenotypic effects from the same locus. Received: 9 October 1997 / Accepted: 29 January 1998     

Ept7 influences estrogen action in the pituitary gland and body weight of rats

Estrogens control many aspects of pituitary gland biology, including regulation of lactotroph homeostasis and synthesis and secretion of prolactin. In rat models, these actions are strain specific and heritable, and multiple quantitative trait loci (QTL) have been mapped that impact the responsiveness of the lactotroph to estrogens. One such QTL, Ept7, was mapped to RNO7 in female progeny generated in an intercross between BN rats, in which the lactotroph population is insensitive to estrogens, and ACI rats, which develop lactotroph hyperplasia/adenoma and associated hyperprolactinemia in response to estrogen treatment. The primary objective of this study was to confirm the existence of Ept7 and to quantify the impact of this QTL on responsiveness of the pituitary gland of female and male rats to 17β-estradiol (E2) and diethylstilbestrol (DES), respectively. Secondary objectives were to determine if Ept7 influences the responsiveness of the male reproductive tract to DES and to identify other discernible phenotypes influenced by Ept7. To achieve these objectives, a congenic rat strain that harbors BN alleles across the Ept7 interval on the genetic background of the ACI strain was generated and characterized to define the effect of administered estrogens on the anterior pituitary gland and male reproductive tissues. Data presented herein indicate Ept7 exerts a marked effect on development of lactotroph hyperplasia in response to estrogen treatment, but does not affect atrophy of the male reproductive tissues in response to hormone treatment. Ept7 was also observed to exert gender specific effects on body weight in young adult rats.     

Early high expression of IP-10 in F344 rats resistant to Sendai virus-induced airway injury

Weanling F344 and BN rats differ markedly in their susceptibility to Sendai virus-induced airway injury. Early gene expression that controls their differences in susceptibility remains poorly understood. In this study we combined suppressive subtractive hybridization and cDNA library array hybridization to identify genes differentially expressed in virus-susceptible BN and virus-resistant F344 rats during the first 3 days after inoculation. Differential expression of selected clones was further verified by quantitative RT-PCR. Seven virus-induced gene segments were identified. Of them, interferon-gamma-inducible protein 10 (IP-10), Mx1, and guanylate-binding protein-2 mRNA abundance in infected F344 rats was 201.5, 188.2, and 281.7% higher, respectively, than that of infected BN rats at 2 days after inoculation. In situ hybridization indicated that virus-induced IP-10 was expressed mainly in airway epithelial cells of F344 rats. Sendai virus infection can directly induce IP-10 expression in rat tracheal epithelial cells in vitro. IP-10 early high expression might contribute to the resistance to virus-induced airway disease in F344 rats by promoting Th1 responses and increasing antiviral activity.     

Increase of estrogen receptor level by thyroxine in estrogen dependent pituitary tumor (MtT/F84) in rats

A rat transplantable pituitary tumor, MtT/F84, grows much faster in E2 treated rats than in normal females, but is much retarded in thyroidectomized rats. Triiodothyronine (T3) administration in a drinking water increased the tumor growth by the dose dependent manner. The tumor contained both estrogen receptor (ER) and T3 receptor. ER levels both in the nuclei and cytosols elevated 2 to 3 times by the T3 administration compared to those of control. E2 administration promotes the growth of MtT/F84 through elevation of nuclear ER level. T3 may directly elevate cellular ER level and thus it may enhance estrogenic actions including the tumor growth.     

Temporal effects of estradiol and diethylstilbestrol on pituitary and plasma prolactin levels in ovariectomized Fischer 344 and Holtzman rats: a comparison of radioimmunoassay and Nb2 lymphoma cell bioassay.

An Nb2 lymphoma cell bioassay (Nb2BA) and a radioimmunoassay (RIA) were used to compare plasma and pituitary levels of prolactin in ovariectomized Fischer 344 (F344) and Holtzman rats treated with either diethylstilbestrol (DES) or estradiol for up to 8 weeks. The objectives were to determine whether there were temporal differences in prolactin responses in strains with different genetic predispositions to estrogen-induced pituitary tumor formation and to determine whether the results of the two assay methods were equivalent. All rats were ovariectomized for 7 days and all except controls received subcutaneous Silastic implants of DES or 17 beta-estradiol and were sacrificed at intervals from 2 days to 8 weeks later. Pituitary content and plasma levels of prolactin were determined by Nb2BA and RIA and the ratio of these measurements was calculated. DES induced a significant increase in pituitary prolactin in F344 rats by 2 days of treatment, as measured by RIA. Pituitary content increased to a peak by Day 4, after which a gradual decline occurred until the end of the experiment. Nb2BA measurements were similar to those obtained by RIA, except at 8 weeks, when the content determined by Nb2BA was significantly higher than the content determined by RIA. When estradiol was given to F344 rats a pattern of increase and subsequent decrease in pituitary content similar to that seen with DES was observed and levels measured by Nb2BA and RIA were essentially equivalent. Plasma levels of prolactin in DES-treated F344 rats increased exponentially through the 8 weeks, and the Nb2BA measurements were significantly greater than levels determined by RIA throughout the treatment period. Estradiol treatment produced a pattern of change in plasma levels of prolactin similar to that observed with DES, except that RIA and Nb2BA measurements were not different. Comparable results were obtained in Holtzman rats, except plasma levels were not increased to the same degree as seen in F344 rats. From these results, we conclude that DES, but not estradiol, can selectively increase the secretion of prolactin that is more bioactive than immunoreactive and that this effect of DES is observed in F344 and Holtzman rats, although F344 rats released more prolactin in response to estrogens than did Holtzman females.     

Quantitative trait loci determining weight reduction of testes and pituitary by diethylstilbesterol in LEXF and FXLE recombinant inbred strain rats.

Increasing exposure to environmental endocrine disruptor, xeno-estrogen, is a serious hazard to male reproductive activity. To explore possible genetic control in susceptibility to xeno-estrogen, the weight reduction of testes induced by the continuous administration of a synthetic estrogen, diethylstilbesterol, were investigated by quantitative trait analysis in LEXF and FXLE recombinant inbred strain rats, consisting of 21 independent strains, 9 of their substrains, parental F344/Stm and LE/Stm strains, and (F344 x LE)F1. For the weight of testes, one highly significant quantitative trait locus (QTL) and one significant QTL were mapped on chromosomes 7 and 1, respectively. The QTL on chromosome 7 is closely associated with c-myc. Pituitary weight and serum prolactin were also variable among recombinant inbred strains, but no QTL was detected for them in this study.     

Radiation attenuates physiological angiogenesis by differential expression of VEGF, Ang-1, tie-2 and Ang-2 in rat brain

The etiology of radiation-induced cerebrovascular rarefaction remains unknown. In the present study, we examined the effect of whole-brain irradiation on endothelial cell (EC) proliferation/apoptosis and expression of various angiogenic factors in rat brain. F344 × BN rats received either whole-brain irradiation (a single dose of 10 Gy γ rays) or sham irradiation and were maintained for 4, 8 and 24 h after irradiation. Double immunofluorescence staining was employed to visualize EC proliferation/apoptosis in brain. The mRNA and protein expression levels of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1), endothelial-specific receptor tyrosine kinase (Tie-2), and Ang-2 in brain were determined by real-time RT-PCR and immunofluorescence staining. A significant reduction in CD31-immunoreactive cells was detected in irradiated rat brains compared with sham-irradiated controls. Whole-brain irradiation significantly suppressed EC proliferation and increased EC apoptosis. In addition, a significant decrease in mRNA and protein expression of VEGF, Ang-1 and Tie-2 was observed in irradiated rat brains. In contrast, whole-brain irradiation significantly upregulated Ang-2 expression in rat brains. The present study provides novel evidence that whole-brain irradiation differentially affects mRNA and protein expression of VEGF, Ang-1, Tie-2 and Ang-2. These changes are closely associated with decreased EC proliferation and increased EC apoptosis in brain.