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.

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.

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.

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.

Quantification of Porcine Follicle-stimulating Hormone Receptor Messenger Ribonucleic Acid by Reverse Transcription competitive Polymerase Chain Reaction

An easy and reliable method was developed for construction and quantification of competitive templates, which shared the same sequence as the amplified target DNA except for a 20 bp insertion in the middle by recombinant polymerase chain reaction (PCR). Among the advantages of competitive PCR is that any predictable or unpredictable variable that affects amplification has the same effect on both target and competitor species and that the final ratio of amplified products reflects exactly the initial targets. The utilization of a thermostable reverse transcriptase in the RT step was proposed to overcome the problem of the efficiency of target cDNA synthesis. In addition, to obtain reliable measurements, it was recommended to perform four PCR with amounts of competitive template flanking the concentration of the target mRNA.

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.

Polymorphism of Follicle-Stimulating Hormone Receptor Gene in Ningxia Tan Sheep

[ Objective] To study the polymorphism of follicle-stimulating hormone receptor （FSHR） gene in Ningxia Tan sheep and thus to provide a theoretical basis for breeding. I Methodl Genotypes of 111 healthy Ningxia Tan sheep were examined by polymerase chain reaction and restriction fragment length polymorphism （PCR-RFLP）. [ResultS] A 306-bp fragment was amplified. The PCR products digested with restriction enzyme Alu I showed polymorphism with three genotypes, L e., GG, CG and CC. The genotypic frequencies of GG, CG and CC were 0.135 1 （ 15 individuals）, 0.666 7 （74 individuals） and 0.198 2 （22 individuals）, respectively. The allele frequencies of G and C were 0.468 5 and 0.531 5, respectively.[ Conclusion] FSHR aene is Dolvmomhic in Ninaxia Tan Sheeo.

Stability of Human Follicle-Stimulating Hormone Receptor mRNA in Stably Transfected Cells

In order to assess the impact of mRNA degradation on steady state levels of follicle stimulating hormone receptor (FSHR) mRNA and on regulation of FSHR gene expression, the stability and half life of FSHR mRNA were determined in transfected cells expressing recombinant FSHR. Time dependent changes in FSHR mRNA content were determined by nuclease protection solution hybridization assay (NPA) or by qualitative reverse transcription competitive polymerase chain reaction (RT PCR) in cultured hFSHR YI cells, cell lines stably transfected with a human FSHR cDNA. FSHR mRNA content remained constant during 8 h control incubations of hFSHR Y1 cells (NPA, 2.9±0.3 μg/mg RNA; RT PCR, 2.7±0.3 μg/mg RNA). Actinomycin D (ActD, 5 μg/ml) inhibited mRNA synthesis, as assessed by incorporation of uridine into total RNA, by 90 % within 1 h in hFSHR Y1 cells. No effect of ActD on cellular morphology or viability was observed. ActD caused a time dependent decrease in FSHR mRNA content in hFSHR Y1 cell lines with a lag time of 1 h. There were no significant differences in the rate of FSHR mRNA degradation between the two methods of mRNA quantification. The half life of hFSHR mRNA was 3.6±0.2 h by NPA and 3.1±0.1 h by RT PCR. The results indicated that degradation of mRNA was an important process in maintenance of steady state expression of the FSHR gene in cells stably expressing recombinant receptor.

Follicle-stimulating Hormone(FSH) Induced Internalization of Porcine FSH Receptor in Cultured Porcine Granulosa Cells and Chinese Hamster Ovary Cells Transfected with Recombinant Porcine FSH Receptor cDNA

In order to study the fate of human follicle-stimulating hormone (FSH) when hormone binds to its receptor, a quick biochemical method that can differentiate between the surface-bound and internalized hormone was used to determine the internalization induced by FSH in cultured both porcine granulosa cells and Chinese hamster ovary (CHO) cells expressing recombinant porcine FSH receptor. The results showed that FSH was slowly internalized, and the internalized radioactivity (acid resistant) reached a peak 10-12 h after addition of 125 I-hFSH. It was suggested that FSHR do not get internalized rapidly under physiological circumstances precisely because the appropriate sequences are absent.

Maturation, proliferation and apoptosis of seminal tubule cells at puberty after administration of estradiol, follicle stimulating hormone or both

Aim： To assess proliferative and apoptotic potential of the seminiferous epithelium cells in relation to Sertoli cell maturation in newborn rats under the influence of estradiol, follicle stimulating hormone （FSH） or both agents given together. Methods： From postnatal day （PND） 5 to 15 male rats were daily injected with 12.5 μg of 1713-estradiol benzoate （EB） or 7.5 IU of human purified FSH （hFSH） or EB ＋ hFSH or solvents （control）. On postnatal day 16, autopsy was performed. Sertoli cell maturation/function was assessed by morphometry. Proliferation of the seminiferous epithelium cells was quantitatively evaluated using immunohistochemical labeling against proliferating cell nuclear antigen and apoptosis using the TUN-EL method. Results： Although EB inhibited Sertoli cell maturation and hFSH was not effective, a pronounced acceleration of Sertoli cell maturation occurred after EB ＋ hFSH. Whereas hFSH stimulated Sertoli cell proliferation, EB or EB ＋ hFSH inhibited Sertoli cell proliferation. All treatments significantly stimulated germ cell proliferation. Apoptosis of Sertoli cells increased 9-fold and germ cells 2-fold after EB, and was not affected by hFSH but was inhibited after EB ＋ hFSH. Conclusion： At puberty, estradiol inhibits Sertoli cell maturation, increases Sertoli and germ cell apoptosis but stimulates germ cell proliferation. Estradiol in synergism with FSH, but neither of the hormones alone, accelerates Sertoli cell maturation associated with an increase in germ cell survival. Estradiol and FSH cooperate to induce seminal tubule maturation and trigger first spermatogenesis.

Influence of leptin on luteinizing hormone and follicle stimulating hormone secreted from cultured rat anterior pituitary cells

BACKGROUND： Leptin may regulate reproductive function via release of hypothalamic neuropeptide Y. However, it is unknown whether this regulatory effect is limited to the hypothalamus. OBJECTIVE： To detect the effect of different dosages of leptin on luteinizing hormone （LH） and follicle stimulating hormone （FSH） secretion from in vitro cultured rat anterior pituitary cells. DESIGN： Contrast study based on cells. SETTING： This study was performed in the Basic Institute of Chengde Medical College, Chengde City, Hebei Province, China from March to June 2007. MATERIALS： Eighteen female Wistar rats of three months of age, weighing 200-220 g, and of clean grade were used. Leptin was provided by Peprotech Company, DMEM culture medium by Invitrogen Company, and the radioimmunological kit by Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. METHODS： Three glandular organs were regarded as one group for culture of anterior pituitary cells. In the control group, saline was added to the culture medium instead of leptin. In the leptin group, leptin was prepared into different concentrations of 1×10^-12, 1×10^-11, 1×10^-9, 1×10^-7, and 1×10^-6 mol/L for stimulation of cultured cells. The culture supernatant was obtained at three hours after additional of saline/leptin. MAIN OUTCOME MEASURES： Contents of LH and FSH were detected by radioimmunology. RESULTS： Following leptin stimulation, LH release increased with increasing concentrations of leptin up to 1×10^-9 mol/L, where LH release peaked. LH release then progressively decreased with increasing leptin concentrations （P 〈 0.01）. LH release in the leptin （1×10^-12, 1×10^-11, 1×10^-7, and 1×10^-6 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. FSH content in the leptin （1×10^-11, 1×10^-9, and 1×10^-7 mol/L） groups was significantly higher than that in the control group （P 〈 0.01）. CONCLUSION： Leptin can directly affect pituitary tissue to promote the secretion of LH and FSH in a dose-dependent manner.

Urinary follicle stimulating hormone can be used as a biomarker to assess male reproductive function

Aim: To develop an algorithm for use in population-based studies to assess testicular function by measurements of totalurinary follicle stimulating hormone (FSH). Methods: Total concentrations of urinary FSH were measured in a groupof 44 men at the University of California, Davis (UCD) and were compared to FSH measurements in serum. On thebasis of these and other published data, a urinary FSH value of >2 ng/mg creatinine (Cr) was selected as the cutoffpoint to identify men with elevated serum FSH ( > 12 IU/L) or low sperm counts ( < 20 million/mL). Results: Thesensitivity and specificity of this algorithm for detecting elevated serum FSH in a group of 58 agricultural workers in thePeople's Republic of China were 100 % and 50 %, respectively. The sensitivity and specificity of this algorithm fordetecting low sperm counts in a population of 105 infertility patients at UCD were 58 % and 76 %, respectively.Conclusion: This test may have particular value in identifying populations with no evidence of testicular toxicity, andin which labor-intensive semen studies may not be feasible.

High expression of follicle stimulating hormone receptor in testicular tissue of idiopathic azoospermic patients with severe spermatogenic defects

Background Follicle stimulating hormone is necessary for normal reproduction in men.The biochemical actions of follicle stimulating hormone result from binding to the follicle stimulating hormone receptor in the plasma membrane of Sertoli cells.Here,we investigated the expression of the follicle stimulating hormone receptor in different testicular histological phenotypes of patients with idiopathic azoospermia.Methods Fifty-seven cases of idiopathic azoospermia were classified into three groups according to the results of testicular biopsy：patients with hypospermatogenesis,patients with maturation arrest,and patients with Sertoli cell-only syndrome.Thirteen azoospermic patients identified by testicular biopsy as being capable of completing spermatogenesis acted as the control group.Immunohistochemistry and real-time quantitative reverse-transcriptase polymerase chain reaction were performed in each case,and the serum hormone level was also measured in all patients.Results The serum follicle stimulating hormone level in patients with Sertoli cell-only syndrome was significantly higher than in patients with hypospermatogenesis,maturation arrest,and complete spermatogenesis （P＜0.01）.The serum follicle stimulating hormone level in patients with maturation arrest was significantly higher than in patients with hypospermatogenesis and complete spermatogenesis （P＜0.05）.There was no difference in serum follicle stimulating hormone levels in patients with hypospermatogenesis and complete spermatogenesis.The follicle stimulating hormone receptor expression level of testicular samples with Sertoli cell-only syndrome was significantly higher than in those with hypospermatogenesis,maturation arrest,and complete spermatogenesis （P＜0.05）,but no significant difference was observed among hypospermatogenesis,maturation arrest,and complete spermatogenesis testicular samples.Conclusions Different serum follicle stimulating hormone levels and follicle stimulating hormone receptor expression were found in the different testicular histology phenotypes in azoospermic patients.Differential follicle stimulating hormone receptor expression in testicular tissue of patients with idiopathic azoospermia may be associated with the degree of spermatogenesis.

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200~ implanted heifers

Background： Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments： non-implanted （n = 5）, Revalor 200 for 28 d （n = 5）, or Revalor 200 for 84 d （n = 6）. Small follicle （1 to 5 mm） granulosa cells were isolated from each pair and incubated with phosphate buffered saline （n = 16） or 100 ng/mL follicle stimulating hormone （n = 16） for 24 h. Results： Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar （P = 0.22） to non-implanted heifers, while medium estradiol concentrations were increased （P 〈 0.10） at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein （P = 0.05） mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 （P 〈 0.10） or 84 d （P 〈 0.05） compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase （P= 0.57） and aromatase （P = 0.23） were demonstrated in implanted or non-implanted heifers. Conclusions： These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an altered capacity to respond to follicle stimulating hormone stimulation. Thus, efforts should be made to avoid the use of implanted heifers to study steroidogenesis in small follicle granulosa cell culture systems.

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.

Antisense Oligodeoxynucleotide Inhibits Expression of Re-com binantPorcine Follicle-Stim ulating Horm one Receptor

To assess the role of follicle stimulating hormone receptor(FSHR) gene expression in regulating expression of FSHR protein in the plasma membrane, the effects of a porcine FSHR cDNA antisense oligodeoxynucleotide (ODN) on FSHR mRNA levels and 125 I FSH binding were determined in Chinese hamster ovary cells expression recombinant porcine FSHR (pFSHR CHO cells). An 18 mer phosphorothioate endcapped antisense ODN that corresponded to the region surrounding the translation initiation codon of the porcine FSHR cDNA was synthesized. An 18 mer nonsense sequence of identical nucleotide composition, which had little homology to known DNA sequences, was synthesized for use as a control. pFSHR CHO cells were cultured in 24 well plates (10 5 cells/well) in the absence or presence of 1 20 μmol/L antisense or nonsense ODN for 24 h and then assayed for porcine FSHR mRNA, using quantitative reverse transcription and competitive polymerase chain reaction, and for 125 I FSH binding activity. Treatment with 10 μmol/L antisense ODN caused a paradoxical increase in porcine FSHR mRNA from 0.89±0.06 to 1.64±0.08 ng/mg total RNA ( P <0.05). Transfection with lipofectamine and 0.33 μmol/L antisense ODN caused an increase in porcine mRNA from 0.95±0.08 to 1.53±0.07 ng/mg total RAN. This was probably due to upregulation of mRNA synthesis resulting from inhibition of porcine FSHR protein translation. The nonsense ODN had no effect on porcine FSHR mRNA. Antisense, but not nonsense, ODN (10 μmol/L) inhibited membrane binding of 125 I FSH by 13.6± 0.8 % ( P <0.05) in 24 h. Treatment of cells with antisense ODN (10 μmol/L) for 48 h resulted in a 76±1.5 % ( P <0.05) inhibition of 125 I FSH binding. In contrast, transfection with lipofectamine and 0.33 μmol/L antisense ODN at 0 h caused a 76.1±1.3 % ( P <0.05) reduction in binding within 24 h. Binding had returned to 52.3±2.3 % ( P < 0.05) of normal by 48 h. These results indicate that an antisense ODN corresponding to the region of the translation start site of the porcine FSHR cDNA is an effective specific inhibitor of porcine FSHR synthesis and that inhibition of receptor synthesis causes a decrease in functional membrane bound FSHR.

Targeted expression of human FSH receptor Asp567Gly mutant mRNA in testis of transgenic mice: role of humanFSH receptor promoter

Aim: To specifically express the Asp567Gly human follicle-stimulating hormone receptor (FSHR) under the control of its promoter to evaluate the phenotypic consequences in the presence of normal pituitary function. Methods: We produced transgenic mice overexpressing the Asp567Gly human FSHR under the control of a 1.5kb 5' flanking region fragment of its promoter. Results: Mice were phenotypically normal and fertile. In males, mRNA could be detected in the testis and the brain, indicating that the 1.5kb promoter fragment drives expression not only in the gonads. The testis weight/body weight ratio and the testosterone levels in transgenic and non-transgenic litter mates were similar. By in situ hybridisation we found that the transgenic FSHR was highly expressed in Sertoli cells, spermatocytes and round spermatids. However, a radioligand receptor assay failed to show a significant difference in total FSHR binding sites in testis homogenates of transgenic and wild type animals, suggesting that the transgenic FSHR is probably not translated into functional receptor protein. Conclusion: A 1.5kb 5 '-region of the human FSHR drives mRNA expression of the transgene in the testis but leads to ectopic expression in germ cells and in the brain. No phenotypic consequences could be documented due to the lack of protein expression.

Association of Thr307Ala and Asn680Ser of Follicle-Stimulating Hormone Receptor Gene Polymorphisms with Gonadotropin Administration during Controlled Ovarian Hyperstimulation

Objective:This study is to investigate the effect of different single-nucleotide polymorphisms of follicle-stimulating hormone receptor(FSHR)gene on gonadotropin(Gn)administration dosage during controlled ovarian hyperstimulation(COH)protocol of in vitro fertilization and embryo transfer.Methods:This retrospective study included 184 Chinese infertile women in Center for Reproduction and Genetics of Suzhou Municipal Hospital from June 2012 to 2014.All of the enrolled patients were homogeneous in some basal characteristics,and they all met the eligibility criteria.Blood tests were conducted on day 3 of menstrual cycle or the day of human chorionic gonadotropin administration for hormonal profile analysis and DNA extraction.DNA sequencing was performed for polymorphism analysis.The participants were classified into threonine(Thr)/Thr,Thr/alanine(Ala),and Ala/Ala groups according to genotype at position 307,and asparagine/asparagine(Asn/Asn),Asn/serine(Ser),and Ser/Ser groups according to genotype at position 680.Logistic regression and correlation analyses were performed to identify the effect of these two polymorphisms on Gn consumption.Results:The frequency of Thr307Ala and Asn680Ser distribution was consistent with Hardy-Weinberg equilibrium(P>0.05).No significant difference was found in age,basal hormone levels for different genotype groups.Logistic regression analysis results revealed that patients with Ser680Ser genotype have a higher risk of requiring a high dose of Gn compared with patients with Asn680Asn genotype,while polymorphism of Thr307 Ala has no such effect.Conclusion:This study suggested that FSHR genotype Asn680Ser would be helpful in determining the dosage of Gn in COH;patients with Ser680Ser genotype may require higher dose of Gn.

The Determination and Evaluation of the Biological Activities for the Commercialization of Recombinant Follicle-Stimulating Hormone in Vitro

Follicle-stimulating hormone (FSH) plays a central role in mammals reproduction, with the actions of FSH mediated by follicle-stimulating hormone receptors (FSHRs) on the surface of target cells. The purposes of this study were to determine and evaluate the biological activities for the commercialization of recombinant follicle-stimulating hormone (rFSH) in vitro through the cellular internalization using cloned 293T-FSHR cell lines as target. Using imaging approaches we have found here that a little fluorescent signal from the surface of the cell transferred to the cytoplasm and accumulated around the nucleus by endocytosis. Compared with the control groups, the commercialization of rFSH have not the significant differences of internalization, but the rFSH have promoted the internalization of the fluorescent, suggested that this detection system might as a protocol for the bioactivity of recombinant therapeutic proteins in vitro.

Analysis of thyroid stimulating hormone receptor gene mutation in children with hyperthyroidism

Objective To explore the characterization of thyroidstimulating hormone receptor ( TSHR) gene mutationalspectrum in children with hyperthyroidism from Guangzhou.Methods Ninety children were diagnosed with hyperthyroidismfrom July 2009 to July 2014 in our institute.Their median age at diagnosis was ( 7. 5 ± 3. 4 )years,and there were 28 males and 62 females. Mutationalanalysis were performed by performing polymerasechain reaction(PCR) and DNA direct sequencing of exon10 of TSHR gene. TSHR gene mutations from 50 unrelatedhealthy children were served as controls. The correlationbetween TSHR gene and hyperthyroidism in childrenwas explored. Results A total of 3 mutations were identifiedin ninety children who were diagnosed with hyperthyroidism,one synonymous mutations(p. V614V),andtwo missense mutations ( p. R707W and p. D727E).