FSH receptor binding inhibitor up-regulates ARID1A and PTEN genes associated with ovarian cancers in mice

Experiments were conducted to determine if the follicle-stimulating hormone (FSH) receptor binding inhibitor (FRBI) impacts the expression levels of AT-rich interactive domain-containing protein 1A (ARID1A) and phosphatase and tensin homolog (PTEN) in ovaries and blood, as well as expressions of follicle-stimulating hormone cognate receptor (FSHR) gene and proteins. Mice in FRBI-10, FRBI-20, FRBI-30, and FRBI-40 groups were intramuscularly injected with 10, 20, 30, and 40 mg FRBI/kg, respectively, for five consecutive days. Western blotting and qRT-PCR were utilized to determine expression levels of ARID1A and PTEN proteins and mRNAs. Serum ARID1A and PTEN concentrations of the FRBI-40 group were higher than the control group (CG) and FSH group (P<0.05). FSHR mRNA levels of FRBI-20, FRBI-30, and FRBI-40 groups were lower than that of CG and FSH groups on day 15 (P<0.05 or P<0.01). Expression levels of FSHR proteins of FRBI-30 and FRBI-40 groups were lower than those of CG and FSH groups (P<0.05). Levels of ARID1A and PTEN proteins of the FRBI-30 group were greater than CG on days 20 and 30 (P<0.05). FRBI doses had significant positive correlations to levels of ARID1A and PTEN proteins. Additionally, ARID1A and PTEN had negative correlations to FSHR mRNAs and proteins. A high dose of FRBI could promote the expression levels of ARID1A and PTEN proteins in ovarian tissues. FRBI increased serum concentrations of ARID1A and PTEN. However, FRBI depressed expression levels of FSHR mRNAs and proteins in mouse ovaries.

Effects of alarelin active immunity on FSHR expressions and bioinformatics of FSHR in ewes / 中国免疫学杂志

Objective:To investigate the effects of GnRH agonist on the expressions of FSHR proteins in pituitary and ovaries in ewes,also to analyse the bioinformatics characteristics of ovine pituitary FSHR.Methods: Forty-two prepubertal ewes (Ovis aries) were randomly assigned to six groups (n=7).The ewes in experimental group EG-Ⅰ,EG-Ⅱand EG-Ⅲwere subcutaneously injected with 200μg,300μg and 400 μg alarelin antigens twice (at day 0 and 14),respectively.Ewes in EG-Ⅳand EG-Ⅴwere injected sub-cutaneously with 200μg and 300μg alarelin antigen four times (at day 0,7,14 and 21),respectively.Ewes in the control group (CG) were subcutaneously injected with 2.0 ml solvent twice (at day 0 and 14).Fluorescence quantitative RT-PCR was used to detect gene expression of FSHR in pituitary glands.The expression of FSHR protein in the ovaries was detected using Western blot.Results:The 2-ΔΔCt values of GnRHR,FSHR and LHR mRNAs in the pituitary gland of EG were lower than that in control group (CG).The 2-ΔΔCt values in EG-Ⅳ( P<0.05 ) and EG-Ⅴ( P<0.05 ) were lower than those in EG-Ⅰand EG-Ⅱ.Expressions of FSHR proteins in EG ovaries increased.Levels of FSHR proteins in EG-Ⅳand EG-Ⅴ( P<0.05 ) were higher than CG.The length of sheep FSHR sequence of nucleotides were 1 091 bp,the homology of FSHR sequences was 100% with that reported in NCBI.The theory isoelectric point , half-life,unstable index,fat index,hydrophobic average were respectively 1.2 h,5.05,47.75,0.836 and 30.61.Conclusion:Alarelin active immunization can inhibit the expression of FSHR mRNA in the pituitary gland ,but increase FSHR expression in ovarian of ewes.FSHR is an unstable hydrophobic protein.

Culture of bovine ovarian follicle wall sections maintained the highly estrogenic profile under basal and chemically defined conditions

Follicle cultures reproduce in vitro the functional features observed in vivo. In a search for an ideal model, we cultured bovine antral follicle wall sections (FWS) in a serum-free defined medium (DM) known to induce 17β-estradiol (E2) production, and in a nondefined medium (NDM) containing serum. Follicles were sectioned and cultured in NDM or DM for 24 or 48 h. Morphological features were determined by light microscopy. Gene expression of steroidogenic enzymes and follicle-stimulating hormone (FSH) receptor were determined by RT-PCR; progesterone (P4) and E2 concentrations in the media were measured by radioimmunoassay. DM, but not NDM, maintained an FWS morphology in vitro that was similar to fresh tissue. DM also induced an increase in the expression of all steroidogenic enzymes, except FSH receptor, but NDM did not. In both DM and NDM, there was a gradual increase in P4 throughout the culture period; however, P4 concentration was significantly higher in NDM. In both media, E2 concentration was increased at 24 h, followed by a decrease at 48 h. The E2:P4 ratio was higher in DM than in NDM. These results suggest that DM maintains morphological structure, upregulates the expression of steroidogenic enzyme genes, and maintains steroid production with a high E2:P4 ratio in FWS cultures.

mRNA Expression of the Regulatory Factors for the Early Folliculogenesis in vitro / 대한불임학회지

OBJECTIVE: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Mullerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. MATERIALS AND METHODS: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in alpha-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. RESULTS: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. CONCLUSION: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

Single nucleotide polymorphisms of follicle-stimulating hormone receptor promoter and their impacts to the promoter activities.

Women of reproductive ages are varies in their responses to exogenous FSH stimulations. The difference of FSHR genotype due to the polymorphisms in exon 10 is one of its significant factors. To know further whether the core promoter of FSHR is also polymorphic and to know whether those polymorphisms influence the promoter activity, we did polymorphism screening of FSHR promoter to 262 women undergoing IVF/ICSI, followed by functional study to know the impact of polymorphisms to the promoter activity. This study indicated that the core promoter of human FSHR is polymorphic. We found five SNPs at positions –29, –37, –114, –123 and –138 in addition to the variety number of adenines. Polymorphism at position –123 significantly decreased the promoter activity, in contrast, polymorphism at position –37 and –138 significantly increased the promoter activity, whereas polymorphism at position –29, –114 and short adenines stretch did not significantly influence the promoter activity. The differences of the promoter activities due to polymorphisms might change the ovarian sensitivity to FSH.

The Effect of Follicle-Stimulating Hormone Receptor (FSHR) Polymorphism on Outcomes of Controlled Ovarian Hyperstimulation (COH) and In-vitro Fertilization and Embryo Transfer (IVF-ET) / 대한불임학회지

OBJECTIVE: To investigate the association of FSH receptor (FSHR) polymorphism at position 680 with outcomes of controlled ovarian hyper-stimulation for IVF-ET in Korean women. Design: Genetic polymorphism analysis. MATERIALS AND METHODS: The FSHR polymorphism was analyzed by PCR-RFLP in 172 ovulatory women below the age of 40 year. Patients with polycystic ovary syndrome, endometriosis, or previous history of ovarian surgery were excluded. RESULTS: Genotype distribution was 41.9% for the Asn/Asn, 47.7% for the Asn/Ser, and 10.5% for the Ser/Ser FSHR genotype group. There was no difference in age of subjects and infertility diagnosis between genotype groups. When the patients were grouped according to their FSHR genotype, the basal levels of FSH (day 3) were significantly different among the three groups (6.0+/-0.3 IU/L (mean+/-SEM), 5.8+/-0.3 IU/L, and 8.6+/-1.2 IU/L for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, p=0.002). The Ser/Ser group showed a higher total doses of gonadotropins required to achieve ovulation induction, and a lower serum estradiol levels at the time of hCG administration compared with other two groups, but the differences were of no statistical significance. The numbers of oocytes retrieved were significantly different among the three groups (8.6+/-0.8, 9.9+/-0.6, and 6.3+/-0.9, for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively, p=0.049). Clinical pregnancy rates were 42.4%, 25.9%, and 29.4% for the Asn/Asn, Asn/Ser, and Ser/Ser groups, respectively. CONCLUSION: Homozygous Ser/Ser genotype of FSHR polymorphism at position 680 was associated with decreased ovarian response to gonadotropin stimulation for IVF-ET.

The expression of follicle-stimulating hormone receptor in ovary and testis.

Follicle-stimulating hormone receptor (FSHR) is exclusively expressed in granulose cells of the ovary and Sertoli cells of the testis. The highly cell-specific of gene expression revealed that transcriptional events unique to these two cell types are responsible for activation of the FSHR gene. Even though its mechanisms are still unclear, several progress regarding the mechanism that control its basal transcription and regulation has been made. It has been identified several important elements that responsible for the transcription of the TATA-less FSHR gene such as: E box element (CACG(A)TG, –124/–119), an inverted GATA (TATC, –88/–85), E2F (TTTCGCG, –45/–39), and regulator element-3 (–197/–171). The functional studies shown that mutations through these regulatory elements significantly decrease the promoter function with greatest impact detected when mutation was done in E-box element. The site-specific CpG methylation within the core promoter seems play an important role in the regulation of rat and mouse FSHR gene expression.

Follicle stimulating hormone receptor gene mutation in Korean women with premature ovarian failure and normal karyotype / 대한산부인과학회잡지

OBJECTIVE: To determine whether the follicle stimulating hormone(FSH) receptor gene mutation (C566T point mutation) is present in Korean women with premature ovarian failure and normal karyotype. METHODS: Genomic deoxyribonucleic acid(DNA) obtained from 40 patients with chromosomally competent premature ovarian failure and from 30 normal fertile women(control group) was amplified by polymerase chain reaction(PCR). PCR products were digested by the enzyme BsmI and polyacrylamide gel(PAG) elctrophoretic patterns of these enzyme-digested products were analyzed. The direct sequencing of PCR products was also performed. RESULTS: All patients with premature ovarian failure and 30 normal control women demonstrated homozygous, normal alleles with 51- and 27- base pairs fragments in PAG elctrophoresis. The absence of C566T point mutation in both group was confirmed by direct DNA sequencing. CONCLUSIONS: A C566T mutation in FSH receptor gene is rare in Korean women with premature ovarian failure and normal karyotype.

Molecular variants of the FSH receptor exon 10 (Thr307Ala; A919G) in premature ovarian failure (POF) women by PCR-SSCP / 대한산부인과학회잡지

OBJECTIVE: This study was performed to determine whether the FSH receptor mutation is present in infertile Korean patients with 46,XX premature ovarian failure (POF) women. METHODS: The variant of FSH receptor exon 10 in thirteen 46, XX idiopathic POF and 4 healthy fertile (control) women were studied. Missense mutation in Exon 10 was detected in POF patients and healthy fertile women by polymerase chain reaction-single stranded conformation polymorphism (PCR-SSCP). RESULTS: The variant types of FSH receptor exon 10 (Thr307Ala; A919G) were found in healthy fertile (control) and POF women. CONCLUSIONS: This mutation may not be specific in POF patients and further study is needed in fertile (control) and POF women.

Analysis of Inactivating Point Mutation of the Follicle Stimulating Hormone Receptor Gene in Korean Infertile Men / 대한남성과학회지

PURPOSE: Follicle stimulating hormone (FSH) is essential for normal gametogenesis. In females FSH is required for ovarian development and follicle maturation whereas in males FSH determines Sertoli cell number and normal spermatogenesis quantitatively and qualitatively. Recently, Tapanainen et al. (1) reported that an anactivating point mutation (C566T) of the FSH receptor gene in males suppressed spermatogenesis but did not cause azoospermia or absolute infertility. To study the significance of the C566T inactivating point mutation in male infertility, we examine the FSH receptor gene in men with azoospermia or oligozoospermia. MATERIALS AND METHODS: Peripheral blood was collected from each patient who had elevated serum FSH. To amplify a suitable segment of the FSHR gene containing nuceotide 566, primer flanking the region was used. And to screen individuals for the C566T mutation, PCR was performed for exon 7 of the FSH receptor gene in 58 patients. RESULTS: The 78-bp fragment containing nucleotide 566 was present in all patient, the PCR product in cleaved into fragments 51-bp and 27-bp by Bsm I digestion. No inactivating point mutations of FSH receptor gene was identified in men with azoospermia or oligozoospermia. CONCLUSIONS: Inactivating point mutation (C566T) of the FSH receptor is not a common cause of male infertility. However we cannot exclude point mutations in other regions of the FSH receptor gene in some patient with azoospermia or oligozoospermia.

STAGE-DEPENDENT EXPRESSION OF ANDROGEN RECEPTOR AND FSH RECEPTOR IN ADULT RAT TESTIS / 解剖学报

Objective To determine the cellular localization and expression pattern of AR and FSHR in adult rat testis must be helpful to understand the action site and mechanisms that T and FSH regulate spermatogenesis. Methods We applied in situ Hybridization to detect the expression of AR and FSHR on adult testis, in which Dig labeled cRNA probe was used to carry out the experiment on frozen sections; at the same time, following the technique of transillumination assisted microdissection we separated seminiferous epithelium into four stages(Ⅱ Ⅵ,Ⅶ Ⅷ,Ⅸ Ⅻ and ⅩⅢ Ⅰ), extracted total RNA and carried out dot hybridization, using ? 32 P labeled cDNA probe, in order to test qualitatively and quantitatively the location of AR and FSHR mRNA and their expression pattern in adult rat testis. Results Our results showed that the positive signal of AR mRNA was located in Sertoli cells and Leydig cells. The signal in Sertoli cells began to appear in Ⅱ Ⅵ stages, strongest in Ⅶ Ⅷ stages and weakest in Ⅸ Ⅰ stages ( P