Mechanism of estrogen in uinduction of rat prolactinoma
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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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To investigate the effect of circulating estrogen level on the outcome of free fat grafting in nude mice.Eighteen female nude mice aged 6-8 weeks (weighing, 20-25 g) were randomly divided into 3 groups ( n=6). The nude mice in the ovariectomized group were treated with ovariectomy. The nude mice in the high estrogen group and the normal estrogen group only made the same incision to enter the peritoneum without ovariectomy. The nude mice in the high estrogen group were given the estradiol (0.2 mg/g) every 3 days for 30 days. The other two groups were given the same amount of PBS every 3 days. At 30 days after operation, the tail vein blood of nude mice in 3 groups were detected by estradiol ELISA kit, and the free fat (0.3 mL) donated by the females was injected into the sub-scalp of nude mice. After 8 weeks of fat grafting, the samples were taken for gross observation and weighing, and the prepared slices were stained with HE staining, CD31-perilipin fluorescence staining, immunohistochemical staining of uncoupling protein 1 (UCP1), and immunofluorescence staining of estrogen receptor α. The diameter of adipocytes and vascular density of adipose tissue were measured. The mRNA expressions of UCP1 and estrogen receptor α were detected by realtime fluorescence quantitative PCR (qRT-PCR).All nude mice survived during experiment. ELISA test showed that the concentration of estradiol significantly decreased in the ovariectomized group and increased in the high estrogen group compared with the normal estrogen group ( P<0.05). At 8 weeks after fat grafting, the graft volume from large to small was ovariectomized group, normal estrogen group, and high estrogen group. There was significant difference in wet weight between the ovariectomized group and high estrogen group ( P<0.05). Section staining showed that compared with the normal estrogen group, the adipocytes in the ovariectomized group were larger, the expression of peri-lipoprotein was weaker, the vascular density decreased, and the expressions of UCP1 was negative, and the estrogen receptor α positive cells reduced. The above observation results in the high estrogen group were contrary to those in the ovariectomized group. There were significant differences in the diameter of adipocytes, the vascular density of adipose tissue, the number of the estrogen receptor α positive cells between groups ( P<0.05). The results of qRT-PCR showed that the mRNA expressions of UCP1 and estrogen receptor α significantly increased in the high estrogen group and decreased in the ovariectomized group compared with the normal estrogen group, and the differences were significant ( P<0.05).The level of circulating estrogen has a significant effect on the outcome of free fat grafting in nude mice. Low estrogen level leads to hypertrophy of graft adipocytes, while high estrogen level leads to the production of a large amount of beige fat and high vascular density in fat grafts, which may be related to the activation of estrogen receptor α on adipocytes.探讨循环雌激素水平对裸鼠颗粒脂肪移植转归的影响。.取 6～8 周龄雌性裸鼠 18 只（体质量 20～25 g），随机分为 3 组（ n=6）。去势组采用卵巢切除术制备去势裸鼠模型，高雌激素组及正常雌激素组仅作相同切口进入腹膜后，不切除卵巢组织。术后高雌激素组每 3 天灌胃雌二醇（0.2 mg/g）1 次，共持续 30 d；其他两组同时间点等量 PBS 灌胃。术后 30 d 取 3 组裸鼠尾静脉血经 ELISA 检测雌二醇浓度后，将接受抽脂术的健康女性捐赠的颗粒脂肪注入裸鼠头皮下（0.3 mL/只）。脂肪移植后 8 周取材大体观察、称重后，制备切片行 HE 染色、CD31-围脂滴蛋白荧光染色、解耦联蛋白 1（uncoupling protein 1，UCP1）免疫组织化学染色和雌激素受体 α 免疫荧光染色，测量脂肪细胞直径及脂肪组织血管密度；实时荧光定量 PCR 检测 UCP1 和雌激素受体 α mRNA 表达。.实验期间裸鼠无死亡。ELISA 检测显示与正常雌激素组相比，去势组雌二醇浓度明显降低，高雌激素组明显升高，差异均有统计学意义（ P<0.05）。脂肪移植 8 周后，移植物体积从大到小依次为去势组、正常雌激素组、高雌激素组，其中去势组移植物湿重显著高于高雌激素组（ P<0.05）。组织学染色显示，与正常雌激素组相比，去势组脂肪细胞增大且围脂滴蛋白表达较弱、血管密度减小，几乎不表达脂肪标记物 UCP1，雌激素受体 α 阳性细胞减少；而高雌激素组上述观察结果与去势组相反；脂肪细胞直径、血管密度及雌激素受体 α 阳性细胞组间比较，差异均有统计学意义（ P<0.05）。实时荧光定量 PCR 检测显示，与正常雌激素组相比，高雌激素组移植物 UCP1 和雌激素受体 α mRNA 相对表达量升高，而去势组均降低；组间比较差异均有统计学意义（ P<0.05）。.体内循环雌激素水平对脂肪移植的转归有显著影响。低雌激素水平导致移植物脂肪细胞肥大，高雌激素水平导致脂肪移植物中生成大量米色脂肪并伴随血管密度增大，其机制可能与升高的循环雌激素水平活化脂肪细胞上雌激素受体 α 相关。.
Nude mouse
Thermogenin
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Changes in prolactin (PRL)‐, growth hormone (GH)‐ and PRL/GH‐containing cells in the anterior pituitary of pre‐ and postnatal male and female rats were determined using immunocytochemistry with double fluorescent antibodies. The pituitary glands from a fetus on Day 20 of gestation and pups on Days 0, 1, 4, 7, 12, and 20 of the postnatal period with sex distinction were monodispersed and subjected to immunocytochemistry. Following immunostaining, the three types of cells described above were counted (i.e., only PRL‐, only GH‐, and both PRL and GH‐positive cells in a visual field of microscope). Anterior pituitaries and blood were obtained from pups on Days 0, 1, 4, 7, 12, and 20 after birth and pooled with sex distinction, and bioactive PRL levels in the anterior pituitary and serum were measured using an Nb 2 rat lymphoma cell bioassay. Double fluorescent‐labeled immunocytochemistry was able to distinguish reliably between PRL‐, GH‐, and PRL/GH‐containing cells. The PRL cells increased rapidly after parturition to 4 days of age and after 12 days of age in both genders. The latter increase was involved in the remarkable increase of PRL/GH‐containing cells, which were first found at term and remained at low levels until 12 days of age. The weight and PRL level of the anterior pituitary increased in both sexes throughout the lactational stage, but the levels in female rats at 20 days of age were significantly greater than those of male rats. Serum PRL concentration remained at low levels except on Day 0. The present results demonstrate that the development of PRL‐containing cells in postnatal rats during the lactational period is activated with two distinct stages, at term to Day 4 and after Day 12, and the development in the latter stage is involved with sexual dimorphism.
Immunostaining
Prolactin cell
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Subcutaneous injection
Mammary tumor
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To observe the effect of acupuncture combined with medication (Rule Granule) on serum prolactin (PRL)，estradiol (E2) and progestone (P) contents and expressions of prolactin receptor (PRLR), estrogen receptor (ER) and progestrone receptor (PR) proteins in mammary gland (MG) tissues of MG hyperplasia (MGH) rats, so as to explore its mechanisms underlying improvement of MGH.A total of 55 female SD rats were randomized into blank control group (n＝10), model group (n＝10), acupuncture group (n＝11), medication group (n＝10), and acupuncture plus medication group (n＝9). The MGH model was established by muscular injection of E2 benzoate(0.5 mg/kg, once daily for 20 days), followed by injection of P (5 mg/kg) into the lateral muscle of the hind-limb, once daily for 5 days, and the rats of the control group were treated by muscular injection of normal saline at the same site and in the same procedures. Acupoint group A composed of bilateral Tianzhong (SI 11), Ganshu (BL 18) and Zusanli (ST 36), and group B composed of Tanzhong (CV 17), and bilateral Wuyi (ST 15) and Hegu (LI 4) were alternatively punctured with filiform needles and stimulated swiftly by twirling the needle in each acupoint for 20－30 sec, once daily for 30 days. Rats of the medication group and acupuncture ＋medication group were treated by gavage of Rule Granule fluid containing Baishao (Paeonia Iactiflora Pall), Danggui (Angelica sinensis), etc. (1.5 mL/100 g for each rat) and those of the other groups treated by gavage of distilled water (1.5 mL/100 g). The treatment was given once daily for continuous 30 d. At the end of the experiments, the rats' abdominal aorta blood was collected for assaying the contents of serum PRL, E2 and P with ELISA, and the pathological changes of breast tissue (the left 2nd pairs) were observed under microscope after sectioning and H．E. staining. The expression of PRLR, ER and PR proteins in the breast tissue was detected by immunohistochemistry.After modeling, the hyperplasia of mammary gland in the model group was obvious; the serum PRL and E2 contents and the immunoactivities of PRLR, ER and PR were significantly increased (P<0.01); and the content of serum P was considerably decreased in the model group in comparison with the blank control group (P<0.01). After the treatment, the hyperplasia of mammary gland was improved; the serum PRL and E2 contents and the expression of PRLR, ER and PR were significantly decreased (P<0.05, P<0.01); and the content of P was notably increased in the acupuncture, medication and acupuncture plus medication groups relevant to the model group (P<0.05，P<0.01). The therapeutic effects of acupuncture plus medication were significantly superior to those of both simple medication and simple acupuncture in down-regulating serum PRL and E2 contents and PRLR, ER and PR immunoactivity, as well as in up-regulating serum P content (P<0.05). No significant differences were found between the medication and acupuncture groups in the above mentioned 3 serum and 3 mammary indexes (P>0.05)．.Acupuncture, Rule Granule, and acupuncture combined with Rule Granule can improve hyperplasia of mammary gland in rats, which may be related to their effects in reducing serum PRL and E2 and breast ER, PR and PRLR expression levels, and in increasing serum P level. The therapeutic effects of acupuncture plus Rule Granule are better than those of simple acupuncture and simple Rule Granule.
Zusanli
Granule (geology)
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To observe the effects of acupuncture combined with Rule granule on breast tissue, prolactin(PRL) and prolactin receptor (PRLR) expression in rats with mammary gland hyperplasia (MGH), and to explore its action mechanism to provide reference for clinical treatment of MGH.Fifty-five female SD rats were randomly divided into a blank group, a model group, an acupuncture group, a Rule granule group and a combination group. Except the blank group, the rats in the remaining groups were treated with combined stimulation of estrogenic and progestational hormone to establish MGH model. After model establishment, the rats in the acupuncture group were treated with acupuncture at Plan A of "Tianzong" (SI 11), "Ganshu" (BL 18), "Zusanli" (ST 36) and Plan B of "Wuyi" (ST 15), "Hegu" (LI 4), "Danzhong" (CV 17). Each plan was selected for one acupuncture treatment, and two plans were used alternately. The rats in the Rule granule group were treated with oral administration of granule, 3 mL per times. The rats in the combination group were treated with the same Rule granule, followed by acupuncture, once a day. After consecutive 30-day treatment, blood sample was collected from abdominal aorta; ELISA method was applied to measure the contents of PRL; the HE slice of mammary gland was observed under light microscope; the SABC immunohistochemical method was applied to measure the positive expression of PRLR.The morphology of breast tissue in the model group was consistent with MGH. Compared with the blank group, the serum PRL and the expression of PRLR were increased significantly in the model group (both P<0.01). Compared with the model group, the hyperplasia of mammary gland in each treatment group was improved, and serum PRL and expression of PRLR were significantly reduced (P<0.05, P<0.01), which were more significant in the combination group (both P<0.05).Acupuncture, Rule granule and its combination could effectively treat MGH, which is likely to reduce the level of serum PRL and inhibit the binding of PRL to PRLR, as a result, the level of E2 is indirectly inhibited, and the hyperplastic mammary gland is recovered. Compared with acupuncture or Rule granule, the combination of both has better overall efficacy.
Zusanli
Prolactin receptor
Granule (geology)
Galactorrhea
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Pituitary tumors were induced in female rats of Wistar strain by administration of estradiol valerate (E2) and the effect of estrogen withdrawal on E2-induced tumors were studied to elucidate the nature of the induced tumors. Ten mg of E2 was administered to 100 rats intraperitoneally once every 2 weeks, totally four times. Experimental animals were sacrificed at 1 week before, 2, 6, 10, and 14 weeks after stopping injection of E2. The incidence of pituitary tumor and hyperplasia (Table 1). Pituitary glands in each animals were examined and were classified into three groups, namely pituitary tumor, hyperplasia, and normal gland according to histological findings. The incidence of pituitary tumors was highest in rats sacrificed 6 weeks after E2 withdrawal. Pituitary tumors became less frequent and hyperplasias and normal glands became more frequent in rats sacrificed thereafter. Pituitary weight (Table 2, Fig. 1) Irrespective of timing of sacrifice, the weight of pituitary gland (mean +/- SE) was 134 +/- 70 mg in the group which were judged as having a pituitary tumor, 38 +/- 17 mg in the hyperplasia group, and 17 +/- 5 mg in the normal group, respectively. Pituitary weight of control rats were 12 +/- 3 mg. The tumor group had a significantly heavier pituitary weight than hyperplasia and normal groups (p less than 0.01). In each group, there was no significant difference of pituitary weights between animals sacrificed at different intervals after E2 withdrawal.(ABSTRACT TRUNCATED AT 250 WORDS)
Pituitary Tumors
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Abstract In a comparative study, female ACI and Sprague-Dawley rats were implanted with pellets containing 5 mg diethylstilbestrol (DES) and 15 mg cholesterol for periods ranging from 2 to 214 days. Strain differences in mammary tumorigenesis, plasma prolactin levels, and gross pituitary responses indicated a desirability for examining the cytology of pituitary glands that were fixed during the course of this study and an immunohistochemical evaluation of their prolactin cell responses. Although plasma prolactin levels were elevated in both strains by 2 days after DES pellet implantation, during the first 10 days of treatment there were no significant increases in pituitary weight in both strains. Pituitary glands from experimental animals of both strains killed on Day 10 exhibited enlarged anti-rat prolactin-binding cells. By Day 28, the pituitary glands of DES-treated ACI rats were significantly enlarged, and a hypertrophic and hyperplastic response of their prolactin cells was detected. From Day 56 to the end of the experiment, gross pituitary tumorigenesis was evident in the ACI but not in the Sprague-Dawley rats. At the microscopic level, adenomatous lesions involving prolactin cell hyperplasia were observed in both strains. Sprague-Dawley and ACI rats exhibited multiple centers of marked vascularization predominated by granular acidophilic cells. These centers were more extensive and larger in the pituitaries of the ACI rats. Two types of immunoreactive prolactin cells were observed in lesioned pituitary glands of both strains: polyhedral, strongly reactive cells which were hypertrophic and more numerous in the ACI rats; and smaller, weakly reactive cells which were numerous in the adenomatous areas of the glands in both strains. It was concluded that the lesions in ACI and Sprague-Dawley females represented different quantitative responses of their prolactin cells to DES treatment.
Prolactin cell
Pituitary Tumors
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Rats with DMBA-induced mammary tumors were given an implant of EB or cholesterol in the ME, or an implant of EB in the cerebral cortex or under the skin. At 25-28 days after implantation, serum prolactin levels in rats with an EB implant in the ME were significantly higher than in the other groups. There were no significant differences among groups in pituitary prolactin concentration, but pituitary prolactin content in rats with an EB implant in the ME was significantly higher than in the other groups. Whereas an implant of EB in the ME resulted in continuous diestrus within a few days, the same dose of EB implanted elsewhere did not disrupt regular estrous cycles. These results indicate that an estrogen implant in the ME can promote pituitary prolactin secretion by direct stimulation of the hypothalamus, pituitary, or both.
Median eminence
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PRL secretion from pituitary lactotrophs was assessed using the reverse hemolytic plaque assay in young (2- to 3-month-old), middle-aged (10- to 12-month-old), and middleaged long term ovariectomized (LT-OVX) rats to investigate whether 1) a change in the percentage of pituitary cells secreting PRL is detectable in middle-aged animals, 2) the amount of PRL secreted per cell changes with age, 3) aging involves a change in responsiveness to TRH and/or dopamine (DA), and 4) LT-OVX can prevent any of these changes. Young and middle-aged rats were OVX for 1 week. LT-OVX rats were OVX at 2–3 months of age and used when they were 10–12 months old. All animals were implanted with Silastic capsules containing estradiol (E2) in sesame oil and killed 3 or 8 days later. Anterior pituitaries were collected, and cells were dispersed and prepared for the reverse hemolytic plaque assay. Three days after E2 was implanted, the percentage of anterior pituitary cells that secrete PRL was higher in middle-aged compared to young rats. LTOVX prevented this increase; the percentage of cells secreting PRL was significantly lower in LT-OVX than in both young and middle-aged rats. Basal secretion of PRL per cell was not different in young compared to middle-aged rats and was significantly lower in LT-OVX than in either young or middle-aged rats. TRH induced similar increases in plaque size in young and middle-aged rats, but had no effect in LT-OVX rats. DA (10-7 M) inhibited plaque size only in LT-OVX rats; however, higher concentrations of DA were equally effective in the three experimental groups. Eight days after E2 was implanted, the percentage of cells that secrete PRL increased in LT-OVX rats, but was still significantly lower than that in middle-aged animals. After 8 days of E2 treatment, PRL release was similar in the three experimental groups under basal conditions. In LT-OVX rats TRH produced a small increase in PRL secretion (30–40%); DA suppressed PRL release in a similar manner in the three groups. These data demonstrate that middle-aged rats exhibit an increase in the percentage of cells secreting PRL without a concomitant detectable change in the amount of PRL released by single cells and/or a change in responsiveness to TRH or DA. Long term estrogen deprivation prevents this age-related change, suppresses responsiveness to TRH, and enhances sensitivity to DA. (Endocrinology124: 90–96,1989)
Silastic
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We have examined testicular and pituitary function in inbred CD-F rats and DBA/2J mice with chronic hyperprolactinemia induced by grafting 4 anterior pituitaries from adult females of the same strain under the kidney capsule. Eight rats were given pituitary isografts and 9 were sham-operated; blood samples were collected at 4-7 week intervals, and the animals were killed 6 months later. One month after surgery, PRL levels in grafted rats were elevated (348 +/- 15 vs. 94 +/- 11 ng/ml; P less than 0.001), LH levels were depressed (16 +/- 3 vs. 59 +/- 9 ng/ml; P less than 0.001), but T levels were not affected (1.0 +/- 0.1 vs. 1.1 +/- 0.2 ng/ml). The elevated PRL levels in grafted animals did not decline during the subsequent 5 months, while LH levels increased slightly, and T levels remained indistinguishable from those in the controls. At the time of autopsy, FSH levels were reduced in grafted rats (230 +/- 40 vs. 501 +/- 108 ng/ml; P less than 0.05). Multiple pituitary isografts did not affect the weight of the testes or the ventral prostate, but increased the weight of the seminal vesicles (P less than 0.001). In 11 mice examined 5.5 months after receiving pituitary isografts, plasma PRL levels were dramatically elevated (330 +/- 35 vs. 27 +/- 2 ng/ml; P less than 0.001), but plasma T levels and testicular weight were not different from those observed in 12 sham-operated controls. The weight of the seminal vesicles in grafted mice was increased (P less than 0.01). In both rats and mice, chronic hyperprolactinemia did not affect plasma testosterone levels or testes weight and increased seminal vesicle weight.
Gonadotropin
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