Mechanism of estrogen in uinduction of rat prolactinoma
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Abstract:
Objective To study the mechanism of estrogen in inducing rat prolactinoma.Methods Adult Wistar rats were divided into two groups at random.Rat in control group(n=5)were subscutaneously implanted with a blank implant.Rats in prolactinoma group(n=5) were implanted with estrogen-containing implants.After 8 weeks,all the animals were sacrificed.Each pituitary gland was weighed and collected for histopathological and immunocytochemical study.Serum prolactin(PRL) levels were measured by RIA.c-fos mRNA levels in pituitary tissue were measured by RT-PCR.Results Pituitary weights,PRL levels and PRL(+)cell counts in prolactinoma group were higher than those in control group(P0.1).Prolactinoma models were developed successfully according to serum PRL level,pituitary weight and immunocytochemical profile.The expression levels of c-fos mRNA in prolactinoma group were obviously higher than those in control group(P0.01).Conclusion Estrogen stimulates c-fos gene expression,which plays an important role in induction of rat prolactinoma.Keywords:
Prolactinoma
Prolactin cell
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Previous observations have shown that a portion of the acute (>12 h) FSH hypersecretion after ovariectomy (OVX) is LHRH independent, thereby suggesting that mechanisms governing the acute FSH hypersecretory response to OVX may reside largely within the anterior pituitary gland. Accordingly, the present studies were conducted to determine whether acute OVX-induced FSH hypersecretion can be elicited in an animal model in which the anterior pituitary gland is isolated from diencephalic chemical signals, and if so, whether the hypersecretion could be abated by the FSH-suppressing protein, follistatin. Adult female rats hypophysectomized (H) 1 week earlier received anterior pituitary grafts (H/G) (one to three glands per rat) under the kidney capsule. In order to increase ovarian secretion of negative feedback effector substances (i.e. estrogen, inhibin), some H/G rats were injected sc with 30 IU PMSG 4-6 days after receiving pituitary transplants, whereas Other rats were given the saline vehicle. Two days later (0830 h), a blood sample was obtained via an indwelling atrial catheter inserted the previous day. H/G rats given saline or PMSG then were further subdivided and either castrated or sham castrated. Additional blood samples were obtained from the catheter, and trunk blood was collected from decapitated rats 24 h after OVX for measurement of serum estradiol and PRL levels. For comparison, H rats not receiving renal pituitary transplants were subdivided into similar experimental groups as the H/G rats. Blood samples were also obtained after sham OVX or OVX of pituitary-intact, 4-day cycling rats on diestrous day 1. Ovariectomy of PMSG-treated H rats receiving either one or three pituitary allografts resulted in a significant (P < 0.01) increase in serum FSH levels by 12 h after OVX followed by a 2- to 3- fold increase in FSH levels by 24 h relative to either the pre- OVX FSH levels measured in this group or the FSH levels measured in PMSG-treated H/G rats 24 h after sham OVX. In contrast, OVX of saline-treated H/G rats failed to elicit FSH hypersecretion. Similarly, FSH hypersecretion was not observed after OVX of saline-or PMSG-treated H rats. Whereas serum LH levels were increased 24 h after OVX of diestrous rats, no such increases were detected 24 h after OVX of any H or H/G rats. In an additional experiment, H rats receiving two pituitary allografts were treated with PMSG and subsequently castrated. Twenty-four hours later, rats were injected iv with either 60 ng purified porcine follistatin or saline. Administration of follistatin suppressed serum FSH levels by approximately 40% 5 h after injection compared to FSH levels measured in saline-treated rats. These data clearly demonstrate that acute OVX-induced FSH hypersecretion can occur from transplanted pituitary tissue, thus providing support for an autonomous component of acute OVXinduced FSH hypersecretion in pituitary-intact rats. Furthermore, the transplanted pituitary tissue retains its responsiveness to FSH regulatory peptides as evidenced by the suppression of FSH release in H/G rats by follistatin. Importantly, in view of the potential existence of local modulators of FSH secretion, the experimental outcome of this study establishes a feasible in vivo model to study paracrine/autocrine regulation of FSH secretion. (Endocrinology128: 1731–1740,1991)
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Objective
To investigate the function of Kiss1 gene and estrogen receptor α gene (ERα gene) in puberty of rats, by detecting the expressions of Kiss1 mRNA and ERα mRNA in the hypothalamus and the serum luteinizing hormone(LH) and estradiol(E2) level of female Sprague-Dawley(SD) rats at various stage of development with Real-time PCR and enzyme-linked immunosorbent assay(ELISA).
Methods
Thirty-five female SD rats of 3 days were weaned on postnatal(PND)22 and then the vaginal opening condition was observed daily.The rats were sacrificed at PND 15(juvenile group, n=19) and PND 35(pubertal group, n=16). The hypothalamus were segregated and the serum were extracted from heart blood.All of the samples were stored at-80 ℃ prepared.Then the mRNA were extracted from the hypothalamus and the cDNA obtained by reverse transcription were tested with real-time PCR.The relative mRNA expression level of Kiss1 gene and ERα gene were calculated.
Results
1.Entire level: it was found that the pubertal group vaginal opening time was (32.1±1.0) days, while the juvenile group was not found with vaginal opening until sacrificed.2.Real-time PCR: the expressions of Kiss1 and ERα gene were significantly increased in pubertal group(Kiss1 gene: 5.39±2.52, ERα gene: 1.57±1.87) compared with juvenile group(Kiss1 gene: 1.06±1.09, ERα gene: 0.59±0.68), and the differences were statistically significant(all P<0.001).3.ELISA: the serum LH and E2 in pubertal group[LH(11.61±0.95) IU/L, E2(167.53±31.09) ng/L]were significantly higher than LH[(5.46±1.89) IU/L]and E2[(58.59±29.96) ng/L]in juvenile group, and the differences were statistically significant(all P<0.001).
Conclusion
Kiss1 gene and ERα gene are involved in the start of the sexual development of female SD rat.
Key words:
Kiss1 gene; Estrogen receptor α; Hypothalamus; Sexual development; Female Sprague-Dawley rat
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Abstract BACKGROUND During formation of prolactin neoplasia, how cells and its structure in adenohypophysis affect prolactin cells should be further studied. Intermediate lobe can be regarded as a driving region to release prolactin (PRL) and may promote formation of prolactin neoplasia in pituitary anterior lobe. OBJECTIVE To observe the effect of diethylstilbestrol (DES) on the expressions of µ and m-calpains in pituitary intermediate lobe of female Wistar rats. DESIGN Observational contrast animal study. SETTING Beijing Neurosurgical Institute. MATERIALS A total of 21 female Wistar rats, 3 weeks old weighing 70 – 80 g were housed with free access to tap water and standard pellet food. They were kept in a CL-grade condition, at (24±1)°C and a humidity of (55±5)%, and with a 12 hours day-night cycle. Caprine anti- µ - and m-calpains antibodies were provided by Santa Cruz Biotechnology, CA, USA; rabbit-anti-PRL antibodies by Dako, Denmark; rabbit-anti-ACTH antibody by Boster Company, Wuhan. METHODS The experiment was carried out in Pathophysiological Department and Animal Laboratory, Beijing Neurosurgical Institute from August 2006 to January 2007. (1) Rats were randomly divided into groups with 7 in each group, including vehicle control group, in which rats were injected intraperitoneally with sun-flower seed oil (1 mL/kg, twice a week) for 16 weeks; DES group, where animals were administered with DES (5 mg/kg, twice a week) for 16 weeks; DES + vehicle control group, in which DES was administered for 12 weeks at the same dose with those in DES group, and then was discontinued and replaced by sun-flower seed oil (1 mL/kg, twice a week) for the following 4 weeks. (2) At 16 weeks later, pituitary tissue was dealt with HE staining and PRL immunohistochemical examination to observe evoke of tumor; meanwhile, immunohistochemical examination was used to observe expression of PRL of pituitary anterior lobe, expressions of µ - and m-calpains of pituitary intermediate lobe and distribution of adrenocorticotropin. MAIN OUTCOME MEASURES (1) Expression of PRL of pituitary anterior lobe, expressions of µ - and m-calpains of pituitary intermediate lobe and distribution of adrenocorticotropin. (2) Morphological observation of pituitary tissue. RESULTS All 21 rats were involved in the final analysis. ? Results of immunohistochemical examination: Morphological changes of neoplasia in DES group were strongly positive to PRL, and this suggested that formation of prolactin adenoma was observed in pituitary tissue. As compared with vehicle control group, expression of adrenocorticotropic hormone (ACTH) was increased in both DES group and DES + vehicle control group. In addition, expressions of µ - and m-calpains in pituitary intermediate lobe were higher in DES group than that in vehicle control group. Otherwise, expressions of m-calpains in pituitary intermediate lobe was decreased in DES + vehicle control group, but expression of µ -calpains was still increased. (2) Morphological observation of pituitary tissue: Gland tubes were orderly arranged in rats in vehicle control group. Anterior pituitary gland in rats of DES group demonstrated an apparent disappearance of gland tubes and a relatively large-scaled vasculature formation, namely the vascular lake lined by tightly arranged endothelial cells. Local integrated tumor cell arrangements were also detected. In addition, the border between the IL and the anterior lobe was locally blurred. The definite tumor-like changes in pituitary tissues were confirmed in 6 of 7 female Wistar rats in DES group, and one spontaneous occurrence of tumor formation was found in vehicle control group. In DES + vehicle control group, DES withdrawal led to the subtile emergence of gland tube cavity, although tumor-like cells still existed in 4 of 7 rats, suggesting occurrence of the tumor regression due to the withdrawal of DES. CONCLUSION A long-term application of DES can enhance the expressions of ubiquitours neutral cysteine protease in pituitary intermediate lobe and this suggests that both of them play a key role in release of hormone and formation of prolactin neoplasia through directly promoting PRL expression and release of neighboring pituitary intermediate lobe.
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Female outbred Sprague-Dawley rats bearing N-nitroso-N-methylurea (NMU)-induced mammary tumors received various endocrine therapies 3 months after the first NMU injection. Rats were divided into 5 groups (15-20 rats/group) and received a 4-week treatment as follows: group 1, controls; group 2, ovariectomized; group 3, 0.5 mg 2-bromoergocryptine (CB-154) injected so twice daily; group 4a, pituitary implant under the kidney capsule; and group 4b, CB-154 injected during the last 2 weeks of the experiment in rats bearing a pituitary implant. Castration of rats with established NMU-induced tumors resulted in a decrease in both tumor number and size, but these parameters again started to increase 3 weeks post castration. CB-154 failed to reduce the tumor number but did arrest the increase in tumor size. In the animals with a pituitary implant, both tumor number and tumor size increased progressively at a greater rate than in control animals, whereas the addition of CB-154 (group 4b) stabilized the tumor growth. Ovariectomy (OVX) resulted in a significant decline of steroid receptor levels. Prolactin (PRL) receptor levels were significantly stimulated by the pituitary implant, and CB-154 prevented this increase. The present studies confirmed that NMU-induced mammary tumors are less hormone-dependent (response to OVX) than 7,12-dimethylbenz[a]anthracene (DMBA)-induced tumors. The role of PRL also appears to be less important, at least for established tumors, for NMU-induced mammary tumors than for DMBA-induced mammary tumors.
Mammary tumor
Pituitary Tumors
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To observe the effect of encircled needling plus electroacupuncture (EA) of the second pair of nipples on hyperplastic mammary glands, serum estradiol (E2), progesterone (P), prolactin (PRL) and testosterone (T) contents, estrogen receptor (ER) expression of the mammary glands in mammary hyperplasia rats.Forty Wistar rats were equally randomized into normal control, model, acupuncture and medication groups. Mammary gland hyperplasia model was established by intramuscular injection of diethylstilbestrol (0.5 mg/kg, once daily for 25 days) and progesterone (4 mg/kg, once daily for 5 days). For rats of the acupuncture group, 4 filiform needles were inserted into the surrounding tissues of the second pair of breasts, respectively, followed by giving the animal with EA stimulation for 30 min. Another one acupuncture needle was inserted into "Danzhong" (CV 17) and retained for 30 min. For rats of the medication group, tamoxifen solution (1.8 mg/kg) was given by gavage. Both medication and EA intervention were given once daily for 30 days. The diameter and height of the second pair of nipples were measured before and on the 10th, 20th and 30th days of the treatment. Serum E2, P, PRL and T levels were assayed by radioimmunoassay, and the tissues of the second pair of nipples were stained with H. E. method. Estrogen receptor immunoactivity of the breast tissue was detected by immunohistochemistry.The diameter and height of the second pair of nipples, serum E2, PRL and T contents, and breast ER protein expression level were significantly higher in the model group than in the normal control group (P<0.05), while serum P content was remarkably lower in the model group than in the normal control group (P<0.05). In comparison with the model group, the diameter and height of the second pair of nipples, serum E2, PRL and T contents, and breast ER protein expression level were considerably lower in both acupuncture and medication groups (P<0.05), and serum P contents were significantly higher in the latter two groups (P<0.05). In addition, H.E. staining showed that the numbers of the lobule, alveolus and ducts of mammary glands were significantly increased, and the expansion of the acinar lumina and ducts was found in the model group. These pathological changes were relatively milder in both acupuncture and medication groups.Encircled needling plus EA stimulation of the surrounding tissues of the nipples can improve the hyperplasic nipples and pathological changes of the breast tissue in mammary gland hyperplasia rats, which is closely associated with its functions in lowering serum estradiol, prolactin, testosterone contents and estrogen receptor protein expression of the breast, and in increasing serum progesterone level.
Dry Needling
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Implanting silastic capsules containing estradiol (E2 into castrated female and “fale” rats 5 days before 7,12-dimethyl benz(a)anthracene (DMBA) injection increased the incidence of mammary tumors from 0% to 44.4% and 30% respectively. However, this E2 potentiating effect on DMBA-induced mammary tumors was not detected in intact female or male rats, but the incidence of mammary tumors was much lower in intact male rats than in intact female rats with or without E2 implants. Most rats receiving E2 implants developed prolactin secreting pituitary tumors within 100 days after implantation. Serum estrogen level of rats bearing E2 implants in different groups was maintained between 118.3 pg/ml and 194.1 pg/ml. These estrogen levels were not significantly different from that of normal cycling rats at proestrus. The pituitary tumors induced by E2 implants secreted huge amounts of prolactin as revealed by enormous levels of PRL in blood, but normal levels of blood FSH and LH were determined. A clear-cut decrement of blood prolactin levels was measured when α-bromoergocriptine was given to female rats with or without pituitary tumors. An unexpected induction of prolactin secreting pituitary tumors in rats by maintaining with silastic implant constant blood proestrus estrogen concentrations was clearly demonstrated.
Silastic
Pituitary Tumors
Mammary tumor
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Female Sprague-Dawley rats aged 50 days received 3 intravenous injections of N-nitroso-N-methylurea, 5 mg/100 g of body weight at 4 weekly intervals. When the first palpable mammary tumours appeared, with an incidence of 50 to 60% after the third injection, rats were randomized into 8 groups: group 1 was treated with 17 beta-estradiol; group 2 received progesterone; group 3 received in association 17 beta-estradiol + progesterone; group 4 was treated with tamoxifen. In group 5, rats received ovine prolactin. In groups 6 and 7, female rats were bred. Group 8, as a control group, received no hormonal treatment. Other control groups of 8 rats aged 140 days were made up of rats receiving 17 beta-estradiol alone (group 9) and rats receiving only ovine prolactin (group 10). The last group was kept without any treatment (group 11). The incidence of mammary tumours was followed in groups 1 to 8. All pituitary glands and mammary tumours were removed and weighed when rats were sacrificed after the last hormonal injection in each group. Light microscopy allowed the anatomopathologist to classify all removed mammary tumours in each group as adenocarcinoma. Light and electron microscopies with all pituitary glands in groups 1 to 11 showed no abnormality. The distribution of the pituitary weights was between 4.0 to 6.8 mg per 100 g of body weight. No adenomas were detected in the pituitary glands of the 5-month-old Sprague-Dawley rats receiving N-nitroso-N-methylurea.
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