Cloning and Sequence Analysis on cDNA of Growth Hormone Releasing Hormone Receptor Gene from Wuzhishan Miniature Pig

[Objective] cDNA of growth hormone releasing hormone （GHRH） receptor gene from Wuzhishan miniature pig was cloned and its sequence was also analyzed. [Method] Using genomic DNA extracted from porcine ear tissues of Wuzhishan miniature pig as the template, three pairs of primers were designed by the reported cDNA sequence of porcine GHRH, and cDNA was also amplified by RT-PCR. After being recovered and purified, PCR products were ligated to pMD18-T and then transformed into Escherichia coli DH5a. The transformation products were analyzed by PCR and double enzyme digestion to screen positive clones, and the positive clones were sequenced after identification in LB liquid medium. [ Result] cDNA of Wuzhishan miniature pig GHRH receptor gene was obtained successfully, and its length was 1 577 bp coding 423 amino acids. BLAST analysis showed that there were only 23 nuoleotides in difference between this fragment and pomine GHRH receptor gene, and its homology was 98%. However, both GHRH receptor genes were constituted by 423 amino acids with the sequence homology of 96%. [ Conclusion] This study provides theoretical basis for further studies on the dwarf mechanism of Wuzhishan miniature pig.

Associations of Gonadotropin-Releasing Hormone Receptor (GnRHR) and Neuropeptide Y(NPY) Genes’Polymorphisms with Egg-Laying Traits in Wenchang Chicken

Single nucleotide polymorphisms （SNP） of chicken gonadotropin-releasing hormone receptor （GnRHR） and neuropeptide Y （NPY） were selected to identify the genotypes of Wenchang （Chinese indigenous breed） chicken with restricton fragment length polymorphisms. The associations of the SNPs with the total egg production （NE）, average days of continual laying （ADCL）, and number of double-yolked eggs （DYE） traits were analyzed. The frequency of restriction enzyme A/a alleles in the population was for GnRHR 0.69 （Bpu1102 Ⅰ A） and 0.31 （Bpu1102 Ⅰ a） and for NPY 0.46 （Dra Ⅰ B） and 0.54 （Dra Ⅰ b）. Trait data from a total of 120 hens, which were purebred introduced from Hainan Province, China from one generation were recorded. Two significant effects of genes＇ marker were found： for GnRHR and number of eggs （dominant; t= 2.67, df= 116） and NPY and number of eggs （additive; t= 1.97, df= 116）. The current research supports the effects of GnRHR and NPY genes on egg-laying traits of chickens.

Thyroid hormones and thyroid hormone receptors: Effects of thyromimetics on reverse cholesterol transport

Reverse cholesterol transport (RCT) is a complex process which transfers cholesterol from peripheral cells to the liver for subsequent elimination from the body via feces. Thyroid hormones (THs) affect growth, develop- ment, and metabolism in almost all tissues. THs exert their actions by binding to thyroid hormone receptors (TRs). There are two major subtypes of TRs, TRα and TRβ, and several isoforms (e.g. TRα1, TRα2, TRβ1, and TRβ2). Activation of TRα1 affects heart rate, whereas activation of TRβ1 has positive effects on lipid and lipoprotein metabolism. Consequently, particular interest has been focused on the development of thyromimetic compounds targeting TRβ1, not only because of their ability to lower plasma cholesterol but also due their ability to stimulate RCT, at least in pre-clinical models. In this review we focus on THs, TRs, and on the effects of TRβ1-modulating thyromimetics on RCT in various animal models and in humans.

The preparation and application of N-terminal 57 amino acid protein of the follicle-stimulating hormone receptor as a candidate male contraceptive vaccine

Follicle-stimulating hormone receptor （FSHR）, which is expressed only on Sertoli cells and plays a key role in spermatogenesis, has been paid attention for its potential in male contraception vaccine research and development. This study introduces a method for the preparation and purification of human FSHR 57-amino acid protein （FSHR-57aa） as well as determination of its immunogenicity and antifertility effect. A recombinant pET-28a（＋）-FSHR-57aa plasmid was constructed and expressed in Escherichia coil strain BL21 StarTM （DE3） and the FSHR-57aa protein was separated and collected by cutting the gel and recovering activity by efficient refolding dialysis. The protein was identified by Western blot and high-performance liquid chromatography analysis with a band of nearly 7 kDa and a purity of 97.4%. Male monkeys were immunized with rhFSHR-57aa protein and a gradual rising of specific serum IgG antibody was found which reached a plateau on day 112 （16 weeks） after the first immunization. After mating of one male with three female monkeys, the pregnancy rate of those mated with males immunized against FSHR-57aa was significantly decreased while the serum hormone levels of testosterone and estradiol were not disturbed in the control or the FSHR-57aa groups. By evaluating pathological changes in testicular histology, we found that the blood-testis barrier remained intact, in spite of some small damage to Sertoli cells. In conclusion, our study demonstrates that the rhFSHR-57aa protein might be a feasible male contraceptive which could affect sperm production without disturbing hormone levels.

Association between Two Polymorphisms of Follicle Stimulating Hormone Receptor Gene and Susceptibility to Polycystic Ovary Syndrome: a Meta-analysis

Objective To investigate the association between two polymorphisms of follicle stimulating hormone receptor （FSHR） gene and polycystic ovary syndrome （PCOS） susceptibility. Methods Case-control studies on relationship of Thr307Ala and Asn680Ser polymorphisms in FSHR gene and PCOS susceptibility were searched from PubMed, ISI web of knowledge, EBSCO, and China National Knowledge Infrastructure （CNKI） databases up to March 21, 2013. The pooled odds ratio （OR） and 95% confidence interval （CO were calculated using fixed- or random-effect model based on heterogeneity test in 5 genotype models analyses. Results A total of 11 studies were included in the Meta-analysis. The random-effect analysis showed Asn680Ser was significantly associated with the reduced susceptibility to PCOS with dominant model （Asn/Asn＋Asn/Ser vs. Ser/Ser, OR=0.83, 95% CI： 0.69-1.00）, recessive model （Asn/Asn vs. Asn/Ser＋ Ser/Ser, OR=0.84, 95% CI： 0.72-0.98）, homozygote comparison （Ash/Ash vs. Ser/Ser, 0R=0.79, 95% CI： 0.63-0.98）, and the allele contrast （Asn vs. Ser, OR=0.87, 95% CI： 0.79-0.97） respectively（P=0.02, I2=56.0%）, being protective factors for PCOS. However, no significant associations were found between Thr307Ala and PCOS. Conclusion There might be a significant association between Asn680Ser polymorphism and PCOS.

Fluoride Exposure,Follicle Stimulating Hormone Receptor Gene Polymorphism and Hypothalamus-pituitary-ovarian Axis Hormones in Chinese Women

The effects of fluoride exposure on thefunctions of reproductive and endocrine systemshave attracted widespread attention in academiccircle nowadays. However, it is unclear whether thegene-environment interaction may modify thesecretion and activity of hypothalamus-pituitary-ovarian （HPO） axis hormones. Thus, the aim of thisstudy was to explore the influence of fluorideexposure and follicle stimulating hormone receptor（FSHR） gene polymorphism on reproductivehormones in Chinese women. A cross sectionalstudy was conducted in seven villages of HenanProvince, China during 2010-2011. A total of 679women aged 18-48 years were recruited throughcluster sampling and divided into three groups, i.e.endemic fluorosis group （EFG）, defluoridationproject group （DFPG）, and control group （CG） basedon the local fluoride concentration in drinkingwater. The serum levels of gonadotropin releasinghormone （GnRH）, follicle stimulating hormone（FSH）, luteinizing hormone （LH）, and estradiol （E2）were determined respectively and the FSHRpolymorphism was detected by real time PCR assay.The results provided the preliminary evidenceindicating the gene-environment interaction onHPO axishormones in women.

Effect of neoadjuvant chemotherapy on the expression of hormone receptors and Ki67 in Chinese breast cancer patients:A retrospective study of 525 patients

This study was designed to investigate the effect of neoadjuvant chemotherapy on the expression of hormone receptors and Ki67 in Chinese female breast cancer patients. The expression of estrogen receptor（ER）, progesterone receptor（PR） and Ki67 among 525 neoadjuvant chemotherapy cases was studied by immunohistochemistry.Differences between specimens made through preoperative core needle biopsy and excised tissue biopsy were observed. The positive rates of ER, PR and Ki67 in core needle biopsy and excised tissue biopsy were 65.3% and 63.2%, 51.0% and 42.6%, 65.6% and 43.4%, respectively. The expression of ER, PR and Ki67 in core needle biopsy and excised tissue biopsy had no statistically significant difference. However, after neoadjuvant chemotherapy, the discordance rates of ER, PR and Ki67 were 15.2%（79/521）, 26.9%（140/520） and 44.8%（225/502）, respectively. The ER, PR and Ki67 status changed from positive to negative in 7.5%（39/521）, 13.3%（69/520） and 21.1%（106/502） of the patients, whereas ER, PR and Ki67 status changed from negative to positive in 7.7%（40/521）, 13.6%（71/520）and 23.7%（119/502） of the patients, respectively. These results showed that the status of some biomarkers changes after neoadjuvant chemotherapy and biomarker status needs to be reexamined to optimize adjuvant systemic therapy and better prognosis assessment.

Cloning and Transcriptional Activity of Follicle-Stimulating Hormone Receptor Promoter in the Jintang Black Goat

[ Objective] To clone follicle-stimulating hormone receptor （FSHR） promoter in the Jintang black goat, study its transcriptional activity, and provide a basis for alternative splicing of FSHR gene. [Method] The total DNA were extracted from the womb of Jintang black goat, and one pair of primers were designed for amplification of FSHR promoter fragments, then the sequences and homology were analyzed. The FSHR promoter fragment was inserted into the pcFSHRB1 expression vector to substitute the CMV promoter and construct the pcFSHRB2 expression vector. The pcFSHRB1 and pcFSHRB2 expression vectors were transformed into HEK293 cells, respectively. Then these cells were collected after 24 and 48 h treatment with 2 mlU/ml follicle-stimulating hormone （FSH）, and the cAMP levels were detected. [Result] The FSHR promoter sequence of Jin- tang black goat had 34.2% homology to that of chicken and 41.6% to that of rat, respectively. The transcription initial site of FSHR was at -576 bp and its upstream sequences contained two TATA-boxes, four CAAT-boxes, one E-box and one Wl-box. After treating for 24 and 48 h, the cAMP levels of pcFSHRB2 were respectively 299.581 3 and 125.528 1 pmol/L; and that of pcFSHRB1 were respectively 120.057 1 and 109.940 7 pmoVL. [Conclusion] The FSHR promoter of Jintang black goat is a typical type 2 eukaryotic promoter, and it is also a strong promoter.

Involvement of chromatin and histone acetylation in the regulation of HIV-LTR by thyroid hormone receptor

The HIV-1 LTR controls the expression of HIV-1 viral genes and thus is critical for viral propagation and pathology. Numerous host factors have been shown to participate in the regulation of the LTR promoter. Among them is the thyroid hormone (T3) receptor (TR). TR has been shown to bind to the critical region of the promoter that contain the NFbB and Sp1 binding sites. Interestingly, earlier transient transfection studies in tissue culture cells have yielded contradicting conclusions on the role of TR in LTR regulation, likely due to the use of different cell types and/or lack of proper chromatin organization. Here, using the frog oocyte as a model system that allows replication-coupled chromatin assembly, mimicking that in somatic cells, we demonstrate that unliganded heterodimers of TR and RXR (9-cis retinoic acid receptor) repress LTR while the addition of T3 relieves the repression and further activates the promoter. More importantly, we show that chromatin and unliganded TR/RXR synergize to repress the promoter in a histone deacetylase-dependent manner.

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders

The female internal sex organs develop from the paramesonephric （Mullerian） duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone （AMH）, which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis （AT）. In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 （AMHR2）, the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction （RT-PCR） and immunohistochemistry were used to detect the receptor＇s mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure.

The adenoviral E1A protein relieves gene repression by receptors in v/vo displaces corepressors and unliganded thyroid hormone

The human adenovirus type 5 early region 1A （E1A） is one of two oncogenes present in the adenovirus genome and functions by interfering with the activities of cellular regulatory proteins. The E1A gene is alternatively spliced to yield five products. Earlier studies have revealed that E1A can regulate the function of thyroid hormone （T3） receptors （TRs）. However, analysis in yeast compared with transfection studies in mammalian cell cultures yields surprisingly different effects. Here, we have examined the effect of E1A on TR function by using the frog oocyte in vivo system, where the effects of E1A can be studied in the context of chromatin. We demonstrate that different isoforms of E1A have distinct effects on TR function. The two longest forms inhibit both the repression by unliganded TR and activation by T3-bound TR. We further show that E1A binds to unliganded TR to displace the endogenous corepressor nuclear receptor corepressor, thus relieving the repression by unliganded TR. On the other hand, in the presence of T3, E1A inhibits gene activation by T3-bound TR indirectly, through a mechanism that requires its binding domain for the general coactivator p300. Taken together, our results thus indicate that E1A affects TR function through distinct mechanisms that are dependent upon the presence or absence of T3.

Distinct expression profiles of transcriptional coactivators for thyroid hormone receptors during Xenopus laevis metamorphosis

The biological effects of thyroid hormone (T3) are mediated by the thyroid hormone receptor (TR). Amphibian metamorphosis is one of the most dramatic processes that are dependent on T3. T3 regulates a series of orchestrated developmental changes, which ultimately result in the conversion of an aquatic herbivorous tadpole to a terrestrial carnivorous frog. T3 is presumed to bind to TRs, which in turn recruit coactivators, leading to gene activation. The best-studied coactivators belong to the p160 or SRC family. Members of this family include SRC1/NCoA-1, SRC2/TIF2/GRIP1, and SRC3/pCIP/ACTR/AIB-1/RAC-3/TRAM-1. These SRCs interact directly with liganded TR and function as adapter molecules to recruit other coactivators such as p300/CBP. Here, we studied the expression patterns of these coactivators during various stages of development. Amongst the coactivators cloned in Xenopus laevis, SRC3 was found to be dramatically upregulated during natural and T3-induced metamorphosis, and SRC2 and p300 are expressed throughout postembryonic development with little change in their expression levels. These results support the view that these coactivators participate in gene regulation by TR during metamorphosis.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

Involvement of Met receptor pathway in aggressive behavior of colorectal cancer cells induced by parathyroid hormone-related peptide

BACKGROUND Parathyroid hormone-related peptide(PTHrP)plays a key role in the development and progression of many tumors.We found that in colorectal cancer(CRC)HCT116 cells,the binding of PTHrP to its receptor PTHR type 1(PTHR1)activates events associated with an aggressive phenotype.In HCT116 cell xenografts,PTHrP modulates the expression of molecular markers linked to tumor progression.Empirical evidence suggests that the Met receptor is involved in the development and evolution of CRC.Based on these data,we hypothesized that the signaling pathway trigged by PTHrP could be involved in the transactivation of Met and consequently in the aggressive behavior of CRC cells.AIM To elucidate the relationship among PTHR1,PTHrP,and Met in CRC models.METHODS For in vitro assays,HCT116 and Caco-2 cells derived from human CRC were incubated in the absence or presence of PTHrP(1-34)(10-8 M).Where indicated,cells were pre-incubated with specific kinase inhibitors or dimethylsulfoxide,the vehicle of the inhibitors.The protein levels were evaluated by Western blot technique.Real-time polymerase chain reaction(RT-qPCR)was carried out to determine the changes in gene expression.Wound healing assay and morpho logical monitoring were performed to evaluate cell migration and changes related to the epithelialmesenchymal transition(EMT),respectively.The number of viable HCT116 cells was counted by trypan blue dye exclusion test to evaluate the effects of irinotecan(CPT-11),oxaliplatin(OXA),or doxorubicin(DOXO)with or without PTHrP.For in vivo tests,HCT116 cell xenografts on 6-wk-old male N:NIH(S)_nu mice received daily intratumoral injections of PTHrP(40μg/kg)in 100μL phosphate-buffered saline(PBS)or the vehicle(PBS)as a control during 20 d.Humanitarian slaughter was carried out and the tumors were removed,weighed,and fixed in a 4%formaldehyde solution for subsequent treatment by immunoassays.To evaluate the expression of molecular markers in human tumor samples,we studied 23 specimens obtained from CRC patients which were treated at the Hospital Interzonal de Graves y Agudos Dr.JoséPenna(Bahía Blanca,Buenos Aires,Argentina)and the Hospital Provincial de Neuquén(Neuquén,Neuquén,Argentina)from January 1990 to December 2007.Seven cases with normal colorectal tissues were assigned to the control group.Tumor tissue samples and clinical histories of patients were analyzed.Paraffin-embedded blocks from primary tumors were reviewed by hematoxylin-eosin staining technique;subsequently,representative histological samples were selected from each patient.From each paraffin block,tumor sections were stained for immunohistochemical detection.The statistical significance of differences was analyzed using proper statistical analysis.The results were considered statistically significant at P<0.05.RESULTS By Western blot analysis and using total Met antibody,we found that PTHrP regulated Met expression in HCT116 cells but not in Caco-2 cells.In HCT116 cells,Met protein levels increased at 30 min(P<0.01)and at 20 h(P<0.01)whereas the levels diminished at 3 min(P<0.05),10 min(P<0.01),and 1 h to 5 h(P<0.01)of PTHrP treatment.Using an active Met antibody,we found that where the protein levels of total Met decreased(3 min,10 min,and 60 min of PTHrP exposure),the status of phosphorylated/activated Met increased(P<0.01)at the same time,suggesting that Met undergoes proteasomal degradation after its phosphorylation/activation by PTHrP.The increment of its protein level after these decreases(at 30 min and 20 h)suggests a modulation of Met expression by PTHrP in order to improve Met levels and this idea is supported by our observation that the cytokine increased Met mRNA levels at least at 15 min in HCT116 cells as revealed by RT-qPCR analysis(P<0.05).We then proceeded to evaluate the signaling pathways that mediate the phosphorylation/activation of Met induced by PTHrP in HCT116 cells.By Western blot technique,we observed that PP1,a specific inhibitor of the activation of the protooncogene protein tyrosine kinase Src,blocked the effect of PTHrP on Met phosphorylation(P<0.05).Furthermore,the selective inhibition of the ERK 1/2 mitogen-activated protein kinase(ERK 1/2 MAPK)using PD98059 and the p38 MAPK using SB203580 diminished the effect of PTHrP on Met phosphorylation/activation(P<0.05).Using SU11274,the specific inhibitor of Met activation,and trypan blue dye exclusion test,Western blot,wound healing assay,and morphological analysis with a microscope,we observed the reversal of cell events induced by PTHrP such as cell proliferation(P<0.05),migration(P<0.05),and the EMT program(P<0.01)in HCT116 cells.Also,PTHrP favored the chemoresistance to CPT-11(P<0.001),OXA(P<0.01),and DOXO(P<0.01)through the Met pathway.Taken together,these findings suggest that Met activated by PTHrP participates in events associated with the aggressive phenotype of CRC cells.By immunohistochemical analysis,we found that PTHrP in HCT116 cell xenografts enhanced the protein expression of Met(0.190±0.014)compared to tumors from control mice(0.110±0.012;P<0.05)and of its own receptor(2.27±0.20)compared to tumors from control mice(1.98±0.14;P<0.01).Finally,assuming that the changes in the expression of PTHrP and its receptor are directly correlated,we investigated the expression of both Met and PTHR1 in biopsies of CRC patients by immunohistochemical analysis.Comparing histologically differentiated tumors with respect to those less differentiated,we found that the labeling intensity for Met and PTHR1 increased and diminished in a gradual manner,respectively(P<0.05).CONCLUSION PTHrP acts through the Met pathway in CRC cells and regulates Met expression in a CRC animal model.More basic and clinical studies are needed to further evaluate the PTHrP/Met relationship.

Growth hormone receptor expression in human primary gastric adenocarcinoma

The aim of this study was to determine the expression of growth hormone receptor（GHR） in patients with pri-mary gastric adenocarcinoma.We investigated 48 specimens of primary gastric adenocarcinoma and their cor-responding normal gastric mucosa.Immunohistochemistry and reverse transcription-polymerase chain reaction（RT-PCR） were used to detect the expression of GHR.Immunohistochemical analyses revealed that GHR was expressed in human primary gastric adenocarcinoma（36/48,75.0%） and appeared to be upregulated,compared to the normal mucosa（28/48,58.3%,P 〈 0.001）.A significant correlation was found between GHR expression and tumor stage（P 〈 0.001） and tumor differentiation（P 〈 0.001）.The average positive rate of ki-67 in GHR-positive tumors was 16.06%,while the positive rate in GHR-negative tumors was 6.17%（P 〈 0.01）.The average apoptosis index（AI） of GHR-positive tumors was 3.36%,which was significantly lower than that（7.33%） of GHR-negative tumors.In addition,27 of 48 cases of tumors had GHR mRNA expression,while only 17 of all 48 cases of normal mucosa did so.Our results indicate that the frequency of GHR was significantly higher in primary gastric adeno-carcinoma than that in normal gastric mucosa.GHR expression was significantly correlated with tumor differen-tiation and tumor grade.This finding supported a possible role of growth hormone in primary gastric adenocarci-noma pathophysiology.

Molecular Characterization of Thyroid Hormone Receptors (TRs) and their Responsiveness to T3 in Microhylafissipes

To explore and enrich the molecular mechanisms of thyroid hormone receptors （TRs） in the metamorphosis of amphibians, the cDNA sequences of TRa and TRβ in Microhyla fissipes were cloned and characterized. TRa was 1 706 bp in length with an open reading frame （ORF） of 1 257 bp encoding a predicted protein of 418 amino acids and TRβ was 1 422 bp with an ORF of 1 122 bp encoding a predicted protein of 373 amino acids. Their protein sequences contained 4 conserved domains of the nuclear receptor superfamily with two highly conserved cysteine-rich zinc fingers in the DNA-binding domain, whereas TRβ was 42 amino acids shorter in its A/B domain than TRot. Highly-conserved sequences and structures indicated their conserved functions during metamorphosis. TRa expression reached peak at 12 h and then decreased from 12 h to 48 h. While dramatically up-regulated TRβ was observed after exposure of T3 within 24 h, and it was down-regulated from 24 h to 48 h. The expression pattern of TRβ is similar to that in the natural metamorphosis. Furthermore, tadpoles treated 24 h also resembled the climax of metamorphosis tadpoles and TRβ expression had higher responsiveness than TRa to T3 in M. fissipes. These results suggest M. fissipes may serve as the model to assay environmental compounds on TH signaling disruption.

Effects of Antisense Oligodeoxynucleotide to Follicle-stimulating Hormone Receptor on the Cell Proliferation and Apoptosis in Cells Derived from Human Ovarian Mucinous Cystadenocarcinoma in Vitro

The human ovarian mucinous cystadenocarcinoma （hOMC） cells were co-cultured with antisense oligodeoxynucleotide （antisense ODN）, nonsense ODN, and follicle-stimulating hormone （FSH） at different concentrations for the purpose of observing the effects of antisense ODN to FSH receptor （FSHR） on the proliferation and apoptosis of cultured hOMC cells in vitro. The inhibitory rates of growth were measured by using MTT method on the 2nd, 4th, 6th, 8th and 10th days after the interference of antisense ODN, nonsense ODN, and FSH, respectively. The apoptotic rates and the cell cycles were determined by means of flow cytometry, the apoptosis indexes were detected by using TUNEL, and the expression of caspase-3 was measured by using SP immunohistochemistry. Compared with that in the control group, the proliferative activity of hOMC cells was increased obviously in FSH groups （P〈0.05 or P〈0.01）, decreased distinctly in antisense ODN groups （P〈0.05 or P〈0.01）, and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could significantly antagonize the FSH-promoted cell proliferative activity （P〈0.01）. Compared with those in the control group, the apoptotic rates and the expression of caspase-3 were dramatically increased in the mid- and high-dose antisense ODN groups （P〈0.05 or P〈0.01）, while the number of cells in G1/G0 phase was significantly decreased and that in S phase distinctly increased （P〈0.01）, There was no change in nonsense ODN groups （P〉0.05）, It was suggested that FSH may improve the development of hOMC cells, However, antisense ODN could inhibit proliferative activity and the FSH-promoted proliferative activity in hOMC cells, at the same time, antisense ODN could inhibit hOMC cell growth by inducing apoptosis.

CORRELATION OF STEROID HORMONE RECEPTORS AND CLINICAL PATHOLOGICAL FEATURES WITH PROGNOSIS OF HUMAN BREAST CANCER

Clinical, pathological features and steroid hormone receptors (SR) including receptors of estrogen (ER), progesterone (PR) and androgen (AR) were observed in 58 cases of breast carcinoma, and related to patient 5- year survival rate through stratification and multivariatc analysis. The results showed that histologic tumor type and grading, lymphnode status, ER value and patient age took more important role in patient survival, and SR, especially, conferred survival advantage in advanced cases with tumor size larger than 2 cm, node involved, or TNM Stage Ⅱ-Ⅲ.

Immunohistochemical Localization and Receptor Expression of Gonadotropin-Releasing Hormone in Pituitary of Guangxi Swamp Buffaloes

[ Objective] To locate gonadotropin-releasing hormone （GnRH） in pituitary of Guangxi swamp buffaloes and to provide a theoretical ba- sis for cloning and sequence analysis of GnRH receptor gene. [ Method] GnRH in pituitary were immunohistochemically stained by avidin biotin complex method. The GnRH expression was analyzed with image system. The GnRH receptor gene was amplified by real-time PCR. [ Result] Many GnRH positive cells were detected in pars distalis of adenohypophysis. GnRH were distributed in cytoplasm but not in nuclei. No positive sig- nal was observed in neurohypophysis. In addition, the GnRH receptor gene, 920 bp in size, was amplified. [ Conclusion] A large number of GnRH and GnRH receptor were found in pars distalis of adenohypophysis, which indicates that anterior pituitary is an important tissue for functions of hypo- thalamus-pituitary-gonadal axis and other endocrine axes.

Reconstruction of HaSNPV with helicoverpa hormone receptor 3

In order to develop a more efficient virus for controlling the cotton bollworm Helicoverpa armigera, Helicoverpa hormone receptor 3 （HHR3）, which is involved in the ecdysteroid regulatory pathway, was used to genetically modify wild HaSNPV. HaSNPV-HHR3 budded virus and occlusion body virus were constructed in three steps： preparation of pFastBacHaPhpP10-HHR3 donor plasmid, transposition of HHR3 into the HaBacHZ8 bacmid, and transfection of HzAM1 cells to get HaSNPV-HHR3 virus.HHR3 was proved to be expressed in the HaSNPV-HHR3 virus infected HzAM1 cells by immunoblotting. Results of bioassay indicated that the body weight of the HaSNPV-HHR3 infected larvae was lower than the larvae infected with wild virus and uninfected normal larvae, which suggests that HaSNPV-HHR3 delayed larval growth.