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Objective To optimize the two-dimensional culture system of mouse preantral follicles, and discuss the effect of follicle stimulating hormone (FSH) on the in vitro development of mouse preantral follicles. Methods The primary follicles (PM follicles, 80-100 μm) and early second follicles (ES follicles, 110-130 μm) harvested from mouse ovaries at day 14 were in vitro cultured with different concentrations of r-FSH culture medium (10 mU/ml and 100 mU/ml). The follicles growth in vitro and the oocytes maturation were observed and recorded. The growth model of antral follicles at day 10 and the levels of estradiol (E2) in the different concentrations of culture medium were examined. The expression levels of FSH receptor (FSHR), steroid hormone synthesis rate-limiting enzyme (3β-hydroxyl steroid dehydrogenase, 3β-HSD), 17α-hydroxylase (CYP17) and aromatase (CYP19) in follicles were detected by Western blotting. Results The cavity rates of PM follicles in 10 mU/ml and 100 mU/ml r-FSH media (0.00%±0.00%, 36.14%±4.02%) and oocyte maturation rates (0.00%±0.00%, 23.54%±7.62%) were obviously lower than the cavity rates of ES follicles (78.63%±4.13%, 92.74%±2.54%) and oocyte maturation rates (48.55%±3.73%, 80.88%±4.02%) with statistical significance (P<0.05). The cavity rates of ES follicles in 100 mU/ml r-FSH media (92.74%±2.54%) and oocyte maturation rates (80.88%±4.02%) were obviously higher than the ES follicles in 10 mU/ml r-FSH media (78.63%±4.13%) and oocyte maturation rates (48.55%±3.73%) with statistical significance (P<0.05). ES follicles in 10 mU/ml r-FSH presented a pattern of tiled growth, while in 100 mU/ml r-FSH presented a stereoscopic spatial growth pattern, which was closer to that of the follicles in vivo. The relative expression level of FSHR in ES follicles was obviously higher than that in PM follicles (1.86±0.32 vs. 1.19±0.28, t=4.94, P<0.05). The expressions of 3β-HSD, CYP17 and CYP19, as well as the secretion of E2 in ES follicles cultured with 100 mU/ml r-FSH were significantly higher than in ES follicles cultured with 10 mU/ml r-FSH. Conclusions FSH can not only change the in vitro development rate, but also change the development pattern of preantral follicles in vitro. The ideal follicle development rate and development mode could be obtained by selecting ES follicles cultured in medium containing 100 mU/ml r-FSH.
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Objective To construct phage display antibody library of artificial mutation to compare with the sequence of the natural phage display antibody library. To scientifically evaluate the quality of the artificial mutation of phage display library, and provide some references for the further transformation of the nanobody. Methods Using random mutation method, NNY fixed-point santuration mutation was performed on combine the follicle-stimulating hormone receptor (FSHR) of human nanobody. The mutant DNA sequence was connected to the vector pMECS to construct the phage display library of VHH06-CDR3 random mutation. By sequencing and analysis of DNA sequences, the diversity of the library and the amino acid distribution of CDR3 were compared between mutation library and the immune library of FSHR. The degree of enrichment of cloning was determined by six rounds of affinity screening. Results According to the NNY mutation rule ,the CDR3 regions with 16 amino acids by random mutations was synthesized and the VHH-CDR3 random mutant phage display library was constructed . The phage display library of VHH06-CDR3 random mutant size was 7.36×108 cfu/ml. Polyclonal and monoclonal phage ELISA showed that after six rounds of screening, the output phage and the combination of FSHR showed obvious enrichment, but there was no clone combined with FSHR. Conclusions Although the VHH06-CDR3 mutant phage display library has sequence diversity, it is not conducive to obtaining target antibodies in affinity screening due to the lack of functional diversity of CDR3.
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AIM:To establish an effective method for purification and culture of human cumulus cells(CCs) in vitro,and to compare the characteristics between CCs and mural granulosa cells(MGCs).METHODS:Follicular fluid and cumulus complex from the patients undergoing intracytoplasmic sperm injection were collected.CCs were mechanically cut from cumulus complex and then directly inoculated on a Petri dish, and MGCs were obtained from follicular fluid through density gradient centrifugation.The expression of follicle stimulating hormone receptor(FSHR)was determined by immunofluorescence.The cell growth curves were measured by CCK-8 assay.The secretion of estrogen was detected by ELISA.RESULTS:After incubated for 24 h, the adherence of CCs was observed.CCs and MGCs had similar growth characteristics and FSHR expression.The similar cell growth curves were observed by CCK-8 assay and the results of ELISA showed that they had comparable secretion of estrogen.CONCLUSION:Direct culture of CCs mechanically cutting from cumulus complex is an effective method.CCs had similar growth characteristics,growth curves and secretion of estro-gen to MGCs cultured in vitro and could be a substitutive source of granulosa cell subsets.
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<p><b>Background</b>In the current society, infertility related to age has become a social problem. The in vitro fertilization (IVF) success rate in women with poor ovarian response (POR) is very low. Dandelion extract T-1 (DE-T1) is an effective component of the extract from the leaves and stems of Taraxacum officinale, which is one of the medicines used in some patients with POR, but its molecular mechanism remains unclear.</p><p><b>Methods</b>Following IVF, ovarian granulosa cells (GCs) of sixty patients were extracted and divided into normal ovarian response (NOR) and POR groups. GCs were cultured in a dose-dependent and time-dependent manner with DE-T1, proliferation of GCs was determined by Cell Counting Kit-8 assay, and mRNA levels of insulin-like growth factor 1 receptor (IGF-1R), luteotropic hormone receptor (LHR), follicle-stimulating hormone receptor (FSHR), LHR, and CYP19A1 (aromatase) were determined by quantitative polymerase chain reaction. Progesterone and estradiol (E2) concentrations were determined by enzyme-linked immunosorbent assay.</p><p><b>Results</b>The cell viability gradually increased with the progressive increase in the DE-T1 concentration. Compared with the control group (without DE-T1), the mRNA expressions of FSHR, LHR, IGF-1R, and CYP19A1 were upregulated after the addition of DE-T1, especially in the 2.5% DE-T1 group (P < 0.01). The expression of IGF-1R was upregulated approximately 25 times (24.97 ± 4.02 times) in the POR group with 2.5% DE-T1. E2 and progesterone levels increased with the increasing DE-T1 concentration. There were highly significant differences in the E2 and progesterone secretion between the NOR and POR groups (P < 0.01).</p><p><b>Conclusion</b>DE-T1 may promote steroid hormone synthesis by promoting GC proliferation and upregulating GC receptor expression, thereby improving ovarian endocrine function.</p>
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Objective To investigate the relationship between OHSS and FSHR gene polymorphism. Methods Two hundred and two women were enrolled in this study. The FSHR gene polymorphisms at position 307 and 680 were detected. Results The distribution of the allele frequency and genotype frequency of the position 680 in FSHR gene were significantly different between women with OHSS or not. No significant differences of the position 307 in FSHR gene were observed. Conclusion Themutation of Asn680Ser in FSHR might be closely related with OHSS.
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Background: Polycystic ovary syndrome (PCOS) is an endocrine disorder and the criteria are specified by common complex genetic hyperandrogenism, oligomenorrhea or amenorrhea and polycystic ovary morphology. It is a leading cause of female infertility. The prevelance of PCOS among reproductive age women has been estimated to be 4-12%. The association between PCOS and FSH receptor (FSHR) polymorphism attracts wide attention. The aim of the present study was to evaluate whether polymorphism of FSHR at Ala307Thr codon is associated with PCOS and with clinical features of PCOS patients in Egypt. Results: PCOS patients (n=64) and control subjects (n=65) in the reproductive age were recruited from the outpatient clinic of Obstetrics and Gynecology Department, Mansoura University, Egypt. The Ala307Thr polymorphism in FSHR, and the frequency of respective genotypes was studied and statistical analysis was performed. We found that the heterozygote Ala/Thr genotype was associated with PCOS (64.1%, OR=2.68, CI=0.97, P= 0.033) compared with controls. Conclusion: The variant of Ala307Thr polymorphism of FSHR was associated with PCOS but it may be related to high total testosterone levels. In addition the FSHR polymorphism was not associated with either luteinizing hormone or follicular stimulating hormone. The present study suggests that the variant of the FSHR gene may act as a causative factor of PCOS in Egyptian women.
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Objective To explore if the expression of follicle-stimulating hormone ( FSH ) and its receptor ( FSHR) exist in the rat submandibular gland tissue , which may provide a theoretic evidence for further studying on adjusting function of the FSH in submaxillary gland .Methods Twenty normal SD rats , with a mean body weight of (200 ±20)g, were adopted to collect out the submandibular gland after abdominal cavity anesthesia .The method of in situ hybridization was employed on cellular localization of FSH and FSHR .RT-PCR was used to detect if FSH and its receptor mRNA exist in the rat submandibular gland .RNA was extracted from the submandibular gland , then FSH and the core cDNA of its receptor gene were obtained with reverse transcription polymerase chain reaction and analyzed on its sequence . Results The epithelial cells of serous acinus and granular convoluted tubule in submaxillary gland of rats had FSH and FSHR mRNA hybridized signals , which were detected in cytoplasm with negative nuclei .Specific bands of FSH cDNA and FSHR cDNA were identical to be 193bp and 413bp respectively.Conclusion FSH and FSHR can be synthesized by the epithelial cells of serous acinus and granular convoluted tubule in submaxillary gland of rats , thus this study further proves that the submandibular gland is the target organ of FSH .
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Objective To sequence follicle stimulating hormone receptro (FSHR) promoter of the ovarian granulocyte and initially research the molecular mechanism of the poor ovarian response. Methods To study the relationship between FSHR promoter mutation of ovarian granulocyte and ovarian respone. The 263 bp DNA fragments before FSHR 5'initiation site in 70 cases of patients with poor ovarian respone and 88 cases of patients with ovarian normal respone who were in the cycle of IVF-ET were sequenced, Results There were 63 cases which occurred 29th site G → A point mutation in 158 women and the mutation rate was 40. 0%. Mutation rate [ 60. 0% ( 42/70 ) ] of 29th site G → A in group of poor ovarian respone was significantly higher(χ2 = 21. 450,P < 0. 01 ) than normal response group [ 23.9% ( 21/88 ) ]. There was no obviously variability ( t = 0. 457, P 0. 05 ) of basic FSH values between two groups [ G/G group was (7.2 ± 2. 3) U/L, G/A & A/A group was (7. 1±2. 0) U/L];there was obviously variability (t = 35. 81 ,P < 0. 05 ) in the number of follicles sinus between two groups ( G/G group was 14. 2±1.3, G/A & A/A group was 4. 5±0. 8 ) ;there was obviously variability ( t = 40. 35, P < 0. 05 ) in the number of ovum pick-up between two groups ( G/G group was 14. 0±1.2, G/A & A/A group was 4. 5±1.1 ) ;there was obviously variability (t =25. 80,P <0.05) of FE2-peak value between two groups [G/G group was (2 865±557) pmol/L, G/A & A/A group was (880±211 ) pmol/L] ;there was obviously variability (t =40. 22 ,P <0. 05) in the number of mature eggs ( G/G group was 13.6±1.2, G/A&A/Agroupwas4.3±0. 9).Conclusion The 29th site of FSHR promoter significantly affect the activity of FSHR promoter. Mutation of G→A can weaken promoter activity, so that ovarian granulocyte poor respone to FSH.
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The purpose of the present research was to investigate the effects of polymorphisms of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) genes, evaluated by polymerase chain reaction-restriction fragment length polymorphism in European-Zebu composite beef heifers from six different breed compositions. The polymorphism site analysis from digestion with HhaI and AluI restriction endonucleases allowed the genotype identification for LHR (TT, CT and CC) and FSHR (GG, CG and CC) genes. A high frequency of heterozygous animals was recorded in all breed compositions for both genes, except in two compositions for LHR. The probability of pregnancy (PP) at first breeding was used to evaluate the polymorphism effect on sexual precocity. The PP was analyzed as a binary trait, with a value of 1 (success) assigned to heifers that were diagnosed pregnant by rectal palpation and a value of 0 (failure) assigned to those that were not pregnant at that time. Heterozygous heifers showed a higher pregnancy rate (67 and 66% for LHR and FSHR genes, respectively), but no significant effects were observed for the genes studied (P = 0.9188 and 0.8831 for LHR and FSHR, respectively) on the PP. These results do not justify the inclusion of LHR and FSHR restriction fragment length polymorphism markers in selection programs for sexual precocity in beef heifers. Nevertheless, these markers make possible the genotype characterization and may be used in additional studies to evaluate the genetic structure in other bovine populations.
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Animals , Male , Female , Cattle/genetics , Crosses, Genetic , Polymorphism, Genetic , Receptors, FSH/genetics , Receptors, LH/genetics , Genotype , Meat , Polymerase Chain Reaction , Reproduction/geneticsABSTRACT
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.
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Objective To investigate relationship between levels of follicle-sitimulating horomone receptor(FSHR) and ovarian response induced by gonadotropin hormone,and whether the FSHR expression is correlated with in vitro fertilization(IVF) outcome.Methods Granulose cells were collected from 43 women receiving IVF-embryo transplantation(IVF-ET).According to the number of oocyte,the women were divided into three groups: low response(15).The expression intensity of FSHR was measured by immunohistochemistry technique.The expression intensity of FSHR on the granular cell,the embryological and clinical outcomes were compared and analyzed. Results The expression of FSHR was significantly different in three groups with the highest in high response group(P0.05).The FSHR level was positively correlated either with the number of oocyte (r=0.719) or with the serum E2 levels(r=0.516,P0.05). Conclusion Ovarian response to gonadotropin hormone stimulation is correlated with the level of FSHR in the granulose cells.The development of follicles may be influenced by it.
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The effects of antisense oligodeoxynucleotide (antisense ODN) to follicle-stimulating hormone receptor (FSHR) and follicle-stimulating hormone (FSH) on the expression of proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF) were studied in primary culture cells derived from human ovarian mucinous cystadenocarcinoma (OMC). The prlmary OMC cells were cultured with the enzyme digestion method, and the expression of pan Keratin protein and FSHR mRNA was detected for identification of the cells. OMC cells were co-cultured with antisense ODN, nonsense ODN and FSH with different concentrations for 48 h and 72 h. The expression of PCNA and VEGF was detected by using SP immunohistochemistry. Compared with that in the control group, the PCNA and VEGF expression was increased obviously in FSH groups (P＜0.05 or P＜ 0.01), while decreased significantly in antisense ODN groups (P＜0. 05 or P＜0.01) and unchanged in nonsense ODN groups, respectively. Meanwhile, antisense ODN could antagonize the increased expression of PCNA and VEGF caused by FSH significantly (P＜0.01). It was suggested that FSH might promotethe development of OMC to some extent. Antisense ODN could inhibit the proliferative activity of OMC cells and the promoting proliferative activity enhanced by FSH.
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Annual changes in and photoperiodic influence oh the weight of gonads, a parameter of gonadal activity, are much smaller in female birds than in males. Effect of season and photoperiod on the follicle-stimulating hormone receptors in the testis or ovary was studied using a subtropical weaver finch. The number of follicle-stimulating hormone binding sites per unit testicular weight showed a peak in the non-breeding phase; while the total number of binding sites per two testes was maximal in the breeding phase and minimal in the regressive phase. In contrast, seasonal changes in follicle-stimulating hormone binding sites in the ovary were less marked. Exposure to short-day during the breeding phase induced marked decreases in the numbers of binding sites per unit testicular weight and per two testes. These numbers markedly increased after transfer to long-day during the non-breeding phase. However, there was no significant effect of short-day or long-day exposure on follicle-stimulating hormone binding sites in the ovary. These results suggest that photoperiod is an effective environmental factor in the regulation of follicle-stimulating hormone receptors in the testis and the effect is manifested by pronounced changes in the testicular weight during annual breeding cycle.
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Objective To observe the localization and distribution of follicle stimulating hormone receptor(FSHR) in the rat submandibular gland and stomach. Methods Immunohistochemical SABC method was used in the experiment. Results The serous glandular cells,granular convoluted epithelial cells and all other duct epithelial cells in the submandibular gland and the parietal cells of gastric gland showed FSHR positive immunoreactivity.The positive substance was distributed in the cytoplasm with negative nuclei.Conclusion The function of rat submandibular gland and gastric gland might be regulated by follicle stimulating hormone(FSH) through FSHR.