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.

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.

Follicle-stimulating hormone autoantibody is involved in idiopathic spermatogenic dysfunction

Aim： To detect the anti-follicle-stimulating hormone （FSH） antibody in idiopathic infertile patients and fertile subjects in order to determine the role of this antibody in patients with spermatogenic dysfunction. Methods： The anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay （ELISA）. The functional and structural integrity of the sperm membrane was evaluated with hypo-osmotic swelling （HOS） test and the ultrastructure of the spermatozoa was investigated by transmission electron microscopy （TEM）. Results： The extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa （swollen） was 45.1% ±3.5% in the FSH antibody-positive group and 59.1% ± 6.2% in the FSH antibody-negative control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the anti- FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive group. Conclusion： These data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and infertility.

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.

Role of follicle-stimulating hormone and estradiol benzoate in recovering spermatogenesis in tamoxifen-injured rats

Objective:To evaluate the effects of follicle-stimulating hormone (FSH) and estradiol benzoate (EB) on the recovery of spermatogenesis, histology, sexual hormones levels and testicular gene expression in testes of tamoxifen-injured rats.Methods:Forty adult rats were divided into eight groups in a factorial arrangement of tamoxifen and hormonal treatments. Half of the groups orally received 0.6 mg/kg tamoxifen, and 30 d later tamoxifen and no-tamoxifen groups (controls) were paired and assigned into four hormonal treatments with daily intramuscular injections for 10 consecutive days: 1 mL saline (control);7.5 IU FSH;12 μg/kg EB;and 7.5 IU FSH+12 μg/kg EB. One day after the last treatment, spermatozoa were recovered from epididymis, blood was processed for sex hormones concentration (testosterone, FSH and luteinizing hormone) and testes were processed for histology and RNA extraction for expression of genes related to apoptosis [caspase 3, inducible nitric oxide synthase (iNOS) and B-cell lymphoma-2 (Bcl-2)].Results: Control groups did not show significant changes in most parameters, but hormonal treatments decreased caspase 3 and iNOS and increased Bcl-2 expression. Tamoxifen significantly decreased counts, motility and viability of spermatozoa, Bcl-2 expression and sex hormones. It increased intertubular space, caspase 3 and iNOS expression, and induced seminiferous tubular atrophy. The hormonal treatments reverted spermatogenesis, hormonal levels and histology compared with controls, however not attaining the same sperm quality as controls.Conclusions:Tamoxifen is clearly detrimental to spermatogenesis and overall testicular structure and function, whereas hormonal therapy with FSH and EB can improve testicular function and revert tamoxifen-induced azoospermia.

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.

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.

Effect of Follicle-Stimulating Hormone Dosage, Seasons and Treatment Frequency on Super-Ovulation in Goats

[ Objective] To explore the effects of FSH （ Follicle-stimulating hormone）, seasons and treatment frequency on super-ovulation in donor goats and to provide necessary data for research about embryo transfer and embryo biotechnology in Yangzhou area. [ Method] Two FSH dosages （240 and 300 I U）, two seasons （April- June and October- December）, and two treatment frequencies （one or two times） were used to induce super-ovulation in goats. [ Result] Both average ovulation point and number of transferable embryos were significantly different between the goats given 240 IU FSH and those given 300 lU FSH （average ovulation point, 10.12 vs 15.55; number of transferable embryos, 8.82 vs 13.15） at the 0.05 level. Both average ovulation point and number of transferable embryos were also significantly different between April -June and October- December （ average ovulation point, 9.05 vs 15.55; number of transferable embryos, 7.05 vs 13.15） at the 0.05 level. Super-ovulation effect was not significantly different between the two treatment frequencies. [ Conduslonl The FSH dosages and seasons have significant impact on super-ovu- lation, but repeat super-ovulation does not have the same impact.

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.

Effect of epidermal growth factor on follicle-stimulating hormone-induced proliferation of granulosa cells from chicken prehierarchical follicles

The development of ovarian follicular cells is controlled by multiple circulating and local hormones and factors, including follicle-stimulating hormone （FSH） and epidermal growth factor （EGF）. In this study, the stagespecific effect of EGF on FSH-induced proliferation of granulosa cells was evaluated in the ovarian follicles of egg-laying chickens. Results showed that EGF and its receptor （EGFR） mRNAs displayed a high expression in granulosa cells from the prehierarchical follicles, including the large white follicle （LWF） and small yellow follicle （SYF）, and thereafter the expression decreased markedly to the stage of the largest preovulatory follicle. SYF represents a turning point of EGF/EGFR mRNA expression during follicle selection. Subsequently the granulosa cells from SYF were cultured to reveal the mediation of EGF in FSH action. Cell proliferation was remarkably increased by treatment with either EGF or FSH （0.1-100 ng/ml）. This result was confirmed by elevated proliferating cell nuclear antigen （PCNA） expression and decreased cell apoptosis. Furthermore, EGF-induced cell proliferation was accompanied by increased mRNA expressions of EGFR, FSH receptor, and the cell cycle-regulating genes （cyclins D1 and El, cyclin-dependent kinases 2 and 6） as well as decreased expression of luteinizing hormone receptor mRNA. However, the EGF or FSH-elicited effect was reversed by simultaneous treatment with an EGFR inhibitor AG1478. In conclusion, EGF and EGFR expressions manifested stage-specific changes during follicular development and EGF mediated FSH-induced cell proliferation and retarded cell differentiation in the prehierarchical follicles. These expressions thus stimulated follicular growth before selection in the egg-laying chicken.

Combination of serum inhibin B and follicle-stimulating hormone levels can not improve the diagnostic accuracy on testicular sperm extraction outcomes in Chinese non-obstructive azoospermic men

Background It is still controversial whether the serum inhibin B level is a superior predictor of the presence of sperm in testicular sperm extraction （TESE） in azoospermic men compared with serum follicle-stimulating hormone （FSH）. In this study, we evaluated the diagnostic accuracy of serum inhibin B levels as a predictor of the outcome of TESE in Chinese non-obstructive azoospermic men and compared it with the traditional marker serum FSH and testicular volumes. Methods Basal values of serum hormone levels, testicular volumes and histological evaluation of 305 Chinese non-obstructive azoospermic men were analyzed. The level of inhibin B was measured using a three-step enzyme-linked immunoassay before sperm extraction, and the diagnostic accuracy of prediction of the outcome of TESE was compared for different markers by the receiver operating characteristics （ROC） curve analysis. Results Testicular sperm was successfully retrieved in 137 of 305 patients （44.9%）. The serum level of inhibin B, the FSH and the testicular volume were significantly different between the successful TESE group and the unsuccessful group. According to the ROC curve analysis, for inhibin B, the cut-off value for discriminating between successful and failed TESE was 28.39 pg/ml （sensitivity 83.5%, specificity 79.1%）. For FSH, the best cut-off value for discriminating was 11.05 pg/ml （sensitivity 83.5%, specificity 74.5%）. The area under the ROC curve of serum inhibin B was similar to that of FSH. Combining the serum inhibin B with FSH levels did not improve the predictive value for successful TESE. Conclusions Serum inhibin B and FSH levels are correlated with spermatogenesis. However, inhibin B is not superior to FSH in predicting the presence of sperm in TESE. And the combination of them does not improve the diagnostic accuracy on TESE outcome.

Follicle-stimulating hormone promotes proliferation of cultured chicken ovarian germ cells through protein kinases A and C activation

The study was conducted to investigate the effects of follicle-stimulating hormone (FSH) on embryonic chicken ovarian germ cell proliferation and its possible involvements of protein kinases A (PKA) and C (PKC) pathways.Ovarian cells were treated with FSH alone or in the presence of forskolin (FRSK),PKA inhibitor (H89),PKC activator (PMA) or inhibitor (H7).The germ cell number was counted from micropictures.The immunocytochemistry of proliferating cell nuclear antigen (PCNA) was applied to identify the proliferating cells.The germ cell labeling index (LI) was determined for cell proliferation.The FSH treatment increased the germ cell number,and this stimulating effect was enhanced by FRSK or PMA,but inhibited by H89 or H7 in a dose-dependent manner.Moreover,the PCNA-LI showed parallel changes with germ cell numbers.This study suggests that FSH may stimulate proliferation of cultured chicken ovarian germ cells by activation of both the PKA and PKC signaling pathways.

Follicle-stimulating hormone increases the intramuscular fat content and expression of lipid biosynthesis genes in chicken breast muscle

Intramuscular fat （IMF） is a crucial factor in the quality of chicken meat. The genetic basis underlying it is complex. Follicle-stimulating hormone （FSH）, well-known as an effector in reproductive tissues, was recently discovered to stimulate abdominal fat accumulation in chicken. The effect of FSH on IMF accumulation and the underlying molecular regulatory mechanisms controlling both IMF and abdominal fat deposition in vivo are largely unknown. In this study, two groups of chickens were treated with chicken FSH or a placebo. The lipid content of breast muscle, abdominal fat volume, and serum concentrations of FSH were examined. Related genes implicated in breast muscle and abdominal fat accumulation were also investigated. Compared to the control group, the triglyceride （TG） content of breast muscle and the percentage of abdominal fat in FSH-treated chickens were significantly increased by 64.9% and 56.5% （P〈0.01）, respectively. The FSH content in the serum of FSH-treated chickens was 2.1 times than that of control chickens （P〈0.01）. Results from quantitative real-time polymerase chain reaction （qRT-PCR） assays showed that relative expression levels of fatty acid synthase （FAS）, lipoprotein lipase （LPL）, diacylglycerol acyltransferase 2 （DGAT2）, adipocyte fatty acid binding protein （A-FABP）, and peroxisome proliferator-activated receptor ~ （PPARy） were significantly upregulated in breast muscle following FSH treatment （P〈0.01）. Treatment with FSH also signifi- cantly increased relative expression levels of FAS, LPL, DGA T2, A-FABP, and PPARy in abdominal fat tissue （P〈0.05） The results of principal component analysis （PCA） for gene expression （breast muscle and abdominal fat） showed that the control and FSH treatment groups were well separated, which indicated the reliability of the data. This study demonstrates that FSH plays an important role in IMF accumulation in female chickens, which likely involves the regulation of biosynthesis genes related to lipid metabolism.

Follicle-stimulating hormone(FSH)levels prior to prostatectomy are not related to long-term oncologic or cardiovascular outcomes for men with prostate cancer

Prior research suggests a link between circulating levels of follicle-stimulating hormone(FSH)and prostate cancer outcomes.FSH levels may also explain some of the observed differences in cardiovascular events among men treated with gonadotropin-releasing hormone(GnRH)antagonists compared to GnRH agonists.This study evaluates the association between preoperative FSH and long-term cardiovascular and oncologic outcomes in a cohort of men with long follow-up after radical prostatectomy.We performed a cohort study utilizing an institutional biobank with annotated clinical data.FSH levels were measured from cryopreserved plasma and compared with sex steroids previously measured from the same samples.Differences in oncologic outcomes between tertiles of FSH levels were compared using adjusted cox regression models.Major adverse cardiovascular events(MACE)were similarly assessed using hospital admission diagnostic codes.A total of 492 patients were included,with a median follow-up of 13.1(interquartile range:8.9–15.9)years.Dehydroepiandrosterone sulfate(DHEA-S)levels,but not other androgens,negatively correlated with FSH levels on linear regression analysis(P=0.03).There was no association between FSH tertile and outcomes of biochemical recurrence,time to castrate-resistant prostate cancer,or time to metastasis.MACEs were identified in 50 patients(10.2%),with a mean time to first event of 8.8 years.No association with FSH tertile and occurrence of MACE was identified.Our results do not suggest that preoperative FSH levels are significantly associated with oncologic outcomes among prostate cancer patients treated with radical prostatectomy,nor do these levels appear to be predictors of long-term cardiovascular risk.

LncRNA-m18as1 competitively binds with miR-18a-5p to regulate follicle-stimulating hormone secretion through the Smad2/3 pathway in rat primary pituitary cells

Long noncoding RNAs(lncRNAs)are expressed in different species and different tissues,and perform different functions,but little is known about their involvement in the synthesis or secretion of follicle-stimulating hormone(FSH).In general,we have revealed lnc RNA-micro RNA(mi RNA)-messenger RNA(m RNA)interactions that may play important roles in rat primary pituitary cells.In this study,a new lncRNA was identified for the first time.First,we analyzed the gene expression of lncRNA-m18as1 in different tissues and different stages by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)and observed the localization of lncRNA-m18as1 with fluorescence in situ hybridization,which indicated that this lncRNA was distributed mainly in the cytoplasm.Next,we used RT-qPCR and enzyme-linked immunosorbent assay(ELISA)to analyze the regulation of FSH synthesis and secretion after overexpression or knockdown of lncRNA-m18as1 and found that lncRNA-m18as1 was positively correlated with FSH synthesis and secretion.In addition,mothers against decapentaplegic homolog 2(Smad2)was highly expressed in our sequencing results.We also screened miR-18a-5p from our sequencing results as a miRNA that may bind to lncRNA-m18as1 and Smad2.We used RNA immunoprecipitation-qPCR(RIP-qPCR)and/or dual luciferase assays to confirm that lncRNA-m18as1 interacted with miR-18a-5p and miR-18a-5p interacted with Smad2.Fluorescence in situ hybridization(FISH)showed that lncRNA-m18as1 and miR-18a-5p were localized mainly in the cytoplasm.Finally,we determined the relationship among lncRNA-m18as1,miR-18a-5p,and the Smad2/3 pathway.Overall,we found that lncRNA-m18as1 acts as a molecular sponge of miR-18a-5p to regulate the synthesis and secretion of FSH through the Smad2/3 pathway.

Effect of Progestin-Primed Ovarian Stimulation Protocol in Infertile Women with Basal Follicle-Stimulating Hormone Levels ≥15 IU/L: A Retrospective Analysis

Objective:To evaluate the efficacy of progestin-primed ovarian stimulation(PPOS)protocol in infertile women with high basal follicle-stimulating hormone(FSH)levels≥15 IU/L.Methods:Patients with high basal FSH levels≥15 IU/L with autologous oocytes from September 2016 to March 2019 were reviewed.Either medroxyprogesterone acetate 4 mg/d or clomiphene citrate(CC)50 mg/d was administered daily from day 3 to the trigger day.When serum FSH levels decreased to≤15.0 IU/L,a low dose of human menopausal gonadotropin(hMG)75/150 IU/d was administered to promote late follicular development.Results:Two hundred and twenty women were retrospectively analyzed in this study.Among them,139 patients were administered with PPOS protocol as the study group,and 81 patients were administered with CC protocol as the control group.The numbers of received oocytes and viable embryos were higher in the study group than those in the control group(1.5±1.2 vs.1.2±0.8 and 0.8±0.8 vs.0.5±0.6,respectively,P<0.05).However,hMG duration and dosage were significantly higher in the study group than those in the control group(4.2±2.7 d vs.1.1±2.3 d and 609.1±424.5 IU vs.140.7±231.3 IU,respectively,P<0.01).Incidence of luteinizing hormone surge and cycle cancellation rate were lower in the study group than those in the control group with statistical difference(2.88%vs.16.05%and 36.50%vs.50.63%,respectively,P<0.05).Conclusions:PPOS protocol can effectively downregulate the endogenous FSH levels.Compared with CC protocol,treatment with PPOS protocol in patients with high basal FSH levels≥15 IU/L could receive more oocytes and more viable embryos.

Clinical Observation on the Treatment of Pre and Postmenopausal Syndromes by Modified Wumei Decoction

Objective:To compare the efficacy of Wumei Decoction in pre and postmenopausal patients and its effect on follicle-stimulating hormone(FSH)and estradiol(E2).Methods:Sixty-four patients who attended the Department of Traditional Chinese Medicine I in Cangzhou City Central Hospital from January 2020 to January 2022 were selected and randomly divided into treatment group and control group,32 cases in each group.The treatment group took modified Wumei Decoction orally,1 dose of water boiled 2 times a day,divided into 2 warm doses;the control group took Livial orally,2.5 mg/times,1 time/day,and the observation cycles were all for 3 months.Kupperman score,FSH,E2,clinical symptoms and clinical efficacy were compared between the two groups before and after treatment.Results:The Kupperman score of the two groups decreased after treatment,and the difference was statistically significant;the total effective rate of the treatment group was higher than that of the control group,and the difference was statistically significant;there was no statistical significance in the comparison of FSH before and after the treatment of the two groups,but the FSH values of the two groups were significantly lower than those before,and the difference was statistically significant;there was no statistically significant difference in the comparison of E2 of the two groups before treatment,and the E2 values of the two groups were higher than those of the control group after the treatment.After the treatment,E2 of the two groups of patients was significantly higher than before,and the difference was statistically significant.After treatment,E2 of the treatment group was higher than that of the control group,and the comparison between the groups was statistically significant.Conclusion:There was no significant difference between modified Wumei Decoction and Livial in lowering follicle-stimulating hormone levels;modified Wumei Decoction was superior in raising oestradiol;and modified Wumei Decoction was relatively effective in improving clinical symptoms.

Studies of GnRH-A Active Immunization Effects on LH and FSH Secretion and Histostructure of the Ovary and Uterus in Rabbits

The objective of the study is to investigate the effects of active immunization against gonadotropin releasing hormone agonist （GnRH-A） on secretion of luteinizing hormone （LH） and follicle-stimulating hormone （FSH） in the pituitary, and to observe the histological structures and development about ovaries and uteri in female rabbits. 24 female rabbits （Oryctolagus cuniculus） were divided randomly into 4 groups （n=6）, namely, experimental group I （EG-I）, experimental II （EG-II）, experimental III （EG-III）, and control group （CG）. Rabbits were subcutaneously injected with 1.0 mL GnRH-A （alarelin） antigen respectively at concentrations of 100, 100 and 50 μg mL-1 respectively, in EG-I, EG-II and EG-III. Alarelin antigen was re-injected in EG-II and EG-III with the same dosage on 20 d. CG was a blank. The ovarian and uterine samples were collected aseptically at the end of the experiment of 70 d. The tissue slices were observed under light and electron microscopes. Serum concentrations of LH and FSH were measured with ELISA. The results showed that serum LH concentrations in EG-II and EG-III reached the peak levels on 50 and 40 d respectively, and LH level in EG-II exceeded other 3 groups on 50 d （P0.05）. FSH level in EG-II was higher than those in EG-I, CG （P0.01） and EG-III （P0.05） on 40 d. GnRH-A could increase the number of primary follicles, enlarge the primary follicle vertical diameter （PFV） and primary follicle transverse diameter （PFT）, and promote growth and maturation of follicles. The endometrial epithelium thickness （EET） and uterine wall thickness （UWT） in three EGs were less than that in CG （P0.05）. GnRH-A can increase the quantities of mitochondrial cristaes, cortex granules in cytoplasm, broaden and lengthen zona pellucidas and microvilli of oocytes. It also enlarged nuclei of ooxytes and mitochondria, thereby it promoted the development of oocytes. Re-injection of 100 μg alarelin antigen enhanced the secretion of LH and FSH. GnRH-A promoted the growth and maturation of ovaries and follicles, suppressed uterine development, and also influenced histostructure of ovaries and uteri in female rabbits.

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.

Auricular acupoint application combined with Fujing prescription treatment for patients with premature ovarian failure

Background:To study the efficacy of Fujing prescription combined with auricular acupoint application in premature ovarian failure and its effect on hormone levels.Methods:From March 2018 to March 2020,100 patients with premature ovarian failure treated in our outpatient clinic were selected and randomly divided into a control group of 50 cases and a treatment group of 50 cases.The control group was treated with Femoston for 28 consecutive days,and the treatment group was treated with not only Femoston,but also Fujing prescription and auricular acupoint application.The patients in the two groups were treated with a 3-month course of treatment.The clinical efficacy,traditional Chinese medicine integral,serum hormone level,immune index and the occurrence of adverse reactions were compared between the two groups.Results:After treatment,the indexes of IgG,IgM and IgA in the treatment group were all increased(P<0.05 for all);compared with the control group,the incidence of adverse reactions in the treatment group was lower(P<0.05).Conclusion:The combination of Fujing prescription and auricular acupoint application in the treatment of premature ovarian failure can improve patients'clinical symptoms significantly,resulting in a decrease in follicle-stimulating hormone levels and an increase in estrogen levels.The combination of Fujing prescription and acupoint application has provided a clinical basis for the TCM treatment of premature ovarian failure.