Expression Of GDNF And Its Receptors In Human Luteinized Granulosa Cells And Regulation Of Gonadotropins On Their MRNA Levels

Neurotrophic factors include neurotrophins, neurokines and glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs). GDNF was purified and characterized in 1993 as a growth factor promoting the survival of various neurons. It has since become evident that GDNF is also expressed in tissues outside the central nervous system and that its overall expression is significantly higher in peripheral organs than in neuronal tissues. GDNF has been shown to regulate ureteric branching during embryonic kidney development. In the testis, GDNF dose-dependently affects spermatogonial stem cells, triggering differentiation at low levels and stimulating self-renewal at higher doses. The trophic effects of GDNF are mediated by a receptor complex consisting of two components, GDNF family receptor (GFR) and the rearranged during transformation (RET) transmembrane tyrosine kinase receptor. All GFLs share the receptor tyrosine kinase RET as their common signaling receptor. The ligand-binding specificity of GFLs is determined by GFRαproteins that have unique binding affinities for each GFL. First of all, GDNF specifically bind to GFRα-1. The complex containing GDNF and GFRα-1, then brings two molecules of RET together, triggering transphosphorylation of specific tyrosine residues in their tyrosine kinase domains and intracellular signaling. RET activates several intracellular signaling cascades, which regulate cell survival, differentiation, proliferation, migration, chemotaxis, branching morphogenesis, neurite outgrowth and synaptic plasticity. Interestingly, a comparison of mRNA levels in 15 organs of mouse by in situ hybridization revealed that GDNF was the most prominently expressed in the ovary. Several reports have demonstrated the presence of GDNFmRNA in the adult murine ovary within the granular layer of developing follicle. A recent report suggests that GDNF stimulates oocyte nuclear and cytoplasmic maturation and cumulus cell expansion within cumulus-oocyte complexes, also enhancing the developmental competence of porcine oocyte. Another reporter suggests potential paracrine roles of GDNF in the promotion of completion of meiosisⅠand the development of early embryos. However, the expression of GDNF and its receptors in human luteinized granulosa cells, to date, and the mechanism of regulation of GDNF have not been investigated. Here, on the basis of other researches about GDNF we investigate the expression of GDNF, GFRa-1, RET in human preovulatory luteinized granulosa cells and treatment of cultured granulosa cells with gonadotropins demonstrate the regulation of gonadotropins on their mRNA levels to explore mechanism of GDNF in follicular development and maturation.PartⅠCulture and identification of luteinized granulosa cells from human ovaryOBJECTIVETo establish a pure and stable experimental cell model, we purify and identify cultured luteinized granulosa cells from human ovary by observation of cell morphology and growth cycle. MATERIALS AND METHODSThe eight subjects recruited in this study were undergoing IVF for tubal factor or male factor reason at the Nanfang Hospital Reproductive Center from May to December in 2009. The research protocol was approved by the University and informed written consent was obtained from all participants. The patients were subjected to controlled ovarian hyperstimulation (COH) accomplished with a modified long luteal suppression protocol in an age from 25 to 30 years, basic FSH<7.5mIU/L, body mass index (BMI) varied from 19kg/m2 to 23kg/m2 and serum estrodiol(E2) concentration<4000pg/L on the day of human chorionic gonadotropin (HCG), without ovulation disorder and have a normal menstrual cycle.Luteinized granulosa cells were isolated from follicular aspiration by gradient centrifugation. Briefly, pooled aspirates were layered onto 45% percoll and centrifuged at 1500r/min×20min. The luteinized granulosa cells removed from the gradient-media interface were washed with PBS twice.2×106/L cells were cultured on 35mm plates at 37℃,5%CO2 with 2ml of culture medium containing 20% female fetal calf serum (FCS) and 1% antibiotic-antimycotic cocktail. After 24 hours the medium was replaced and washed by PBS to remove cellular debris and suspended cells. Cell morphology and viability were observed every 24 hours and taken photos at the same time.Luteinized granulosa cells identification:The 2ml of purified suspension of human granulosa cells was dropped onto the polylysine treated glass slides and put into incubator for 24 hours. Then cell samples fixed in 4% paraformaldehyde were processed for paraffin embedding about 30 minutes. After that these samples were incubated with the primary rabbit polyclonal antibodies against FSHR (diluted:1:50). Slide incubated with the secondary antibody alone served as negative controls. Images were captured by an image analysis computer system. RESULTS1. Clinical characteristics of eight patientsThere were eight patients recruited in this study and the average age was 26.4±1.2 years and BMI was (21.3±1.3) kg/m2, basic FSH level was (6.8±0.5)mIU/mL. The average serum E2 level on the day of HCG was (1493.2±755.1)pg/ml.2. Primary human luteinized granulosa cell cultureHuman luteinized granulosa cells were slowly growing under the culture contition. These cell aggregates in culture attached to culture dish within 24 hours and maintained a healthy granular appearance throughout the period of culture in contrast to the spherical shape when seeded. A few erythrocytes were visible in the initial cell culture and loosely attached to the bottom of dish but did not persist which were were totally displaced at 24 hours as judged by microscopy.The generation of pseudopodia increased during the subsequent 24 hours. At 48 hours, the monolayer cells appeared to be widespread and multiple interconnections between the cells were noted. The generation of pseudopodia seemed not to proceed after 72 hours of incubation. Cells were flattened and had a fibroblast-like appearance with stress fibres at 96 hours and part of granular material dispeared within a few granulosa cells. At 120 hours, part of granulosa cells were seemed to typical luteal phenotype with hypertrophic cell bodies, more granular material and short pseudopodia. Generally, cell death was observed by microscopy at 216 hours with attached cells suspending. In the culture of 15 days, all granulosa cells were generally smaller and had either irregular or elongated forms resembling fibroblasts which were different from the typical granulosa cell morphology.3. Identification of human luteinized granulosa cellFSHR was used for identification of granulosa cells specifically as it present exclusively on granulosa cells of ovarian follicles in females. The red-brown staining indicated FSHR protein expression in the luteinized granulosa cells (70%-80% cytoplasmic without nuclear staining).CONCLUSION1. Density gradient centrifugation with Percoll is used widely in granulosa cell purification and erythrocytes are removed effectively as judged by microscopy.2. In the culture medium of RPMI-1640 containing 20% female fetal calf serum (FCS) and 1% antibiotic-antimycotic cocktail, since degenerative changes did not been observed until 96 hours, primary granulosa cells were appropriate for experiment in vitro before 96 hours.3. By identification, the purity of human luteinized granulosa cell in culture is up to 80% and appropriate for experiments in vitro.PartⅡExpression of GDNF,GFR-1,Ret in human luteinized granulosa cellsOBJECTIVETo investigation the expression of GDNF and its receptors, GFRα-1 and Ret in human luteinized granulosa cells at the protein levels by immunocytochemistry.MATERIALs AND METHODSThe fiftheen subjects recruited in this study were undergoing IVF for tubal factor or male factor reason at the Nanfang Hospital Reproductive Center. The research protocol was approved by the University and informed written consent was obtained from all participants. The patients were subjected to COH accomplished with a modified long luteal suppression protocol in an age from 25 to 30 years, basic FSH<7.5mIU/L, BMI varied from 19kg/m2 to 23kg/m2 and serum E2<4000pg/L on the day of HCG, without ovulation disorder and have a normal menstrual cycle.After purification by density gradient centrifugation with 45% Percoll, the 2ml of suspension of human granulosa cells was dropped onto the polylysine treated glass slides and put into incubator for 24 hours. Then cell samples fixed in 4% paraformaldehyde were processed for paraffin embedding about 30 minutes. After that these samples were incubated with the primary antibodies (diluted:1:200,1:200, not diluted) as follows:rabbit polyclonal antibodies against GDNF and GFRα-1, mouse monoclonal antibodies against Ret. Slide incubated with the secondary antibody alone served as negative controls. Images were captured by an image analysis computer system.RESULTS1. Clinical characteristics of fifteen patientsThere were fifteen patients recruited in this study and the average age was 26.7±2.0 years and BMI was (21.5±1.2)kg/m2, basic FSH level was (6.7±0.6) mIU/mL. The average serum E2 level on the day of HCG was (2009.6±850.7)pg/ml.2. The characteristics cellular localization of GDNF in luteinized granulosa cells included full cytoplasmic staining in 100% and nuclear staining in 97%.3. The characteristics cellular localization of GFRa-1 in luteinized granulosa cells included full cytoplasmic staining in 100% and nuclear staining in 55%.4. The characteristics cellular localization of Ret in luteinized granulosa cells included full cytoplasmic staining in 47% and nuclear staining in 17%.CONCLUSIONThe presence of the GDNF and its receptors in human preovulatory luteinized granulosa cells suggest that GDNF may act as an important autocrine/paracrine regulator during follicular development and maturation process. PartⅢRegulation of gonadotropins on GDNF,GFR-1,RET mRNA levels in human luteinized granulosa cellsOBJECTIVETo explore whether FSH, human menopausal gonadotropin (HMG) may regulate the mRNA levels of GDNF and its receptors by treatment of cell culture with gonadotropins. This study is noteworthy and may suggest the mechanism of GDNF in follicular development and maturation process.MATERIALS AND METHODSSamples from five patients were recruited in this study by using random blocks design. Luteinized granulosa cells from every one participant were divided into four groups randomly.2×105 cells were cultured on 35mm culture dish for 24 hours, then rinsed two times with PBS. RPMI-1640 was added with the experimental treatment of culture containing HMG5IU/L (5IU/L group), HMG10IU/L (10IU/L group), FSH 10IU/L (FSH group) respectively and without gonadotropins as blank control group. For quantitative real-time RT-PCR to detect transcripts of GDNF, GFRa-1 and RET by SYBR Green staining, human luteinized granulosa cells were collected at 48 hours after treatment. Each result was repeated at least three times.SPSS 13.0 statistical package was used for date analysis. Relative mRNA level data was expressed by mean±standard deviation and analyzed statistically by using two-way classification ANOVA for multifactorial analysis of vatiance. And data was further analyzed using the Student-Newman-Keuls test for multiple comparisons to determine statistical differences between groups. All values were two-tailed, and P<0.05 was considered statistically.RESULTS1. Clinical characteristics of five patientsThere were five patients recruited in this study and the average age was 27.2±1.9 years and BMI was (21.7±0.7)kg/m2, basic FSH level was (6.8±0.5)mIU/mL. The average serum E2 level on the day of HCG was (1892.2±396.4)pg/ml.2. Relative GDNFmRNA levelsExpression of GDNFmRNA are significantly differences between 5IU/Lgroup, 10IU/Lgroup, FSH group, and control group(F=22.308, P<0.01). GDNFmRNA was enhanced up to 4.37 fold by HMG at concentrations of 10IU/L and 2.76 fold by HMG at concentrations of 5IU/L compared to blank control group significantly. Block design was effective as statistical significances between patients (F=6.283, P=0.006). Stimulation of GDNFmRNA by HMG was demonstrated to be dose dependent by further analysis using the SNK test for multiple comparisons. However, FSH failed to stimulate GDNFmRNA by cultured luteinized granulosa cells at the concentration of 10IU/L.2. Relative GFRα-1mRNA levelsExpression of GFRα-1mRNA are significantly differences between 5IU/Lgroup, lOIU/Lgroup, FSH group, and control group(F= 18.985, P<0.01). GFRα-1mRNA was enhanced up to 4.86 fold by HMG at concentrations of 10IU/L and 3.23 fold by HMG at concentrations of 5IU/L compared to blank control group significantly. Block design was effective as statistical significances between patients (F=4.568, P=0.018). Stimulation of GFRα-1mRNA by HMG was demonstrated to be dose dependent by further analysis using the SNK test for multiple comparisons. However, FSH failed to stimulate GFRα-1mRNA by cultured luteinized granulosa cells at the concentration of 10IU/L3. Relative RETmRNA levelsExpression of RETmRNA are not significantly different between the four groups. 10IU/L FSH,10IU/L HMG and 5IU/L HMG failed to stimulate RETmRNA by cultured luteinized granulosa cells. CONCLUSION1. FSH failed to stimulate GDNFmRNA, GFRα-1mRNA, and RETmRNA by treatment of culture containing 10IU/L FSH. It suggests that FSH at the concentration of 10IU/L can not up-regulate their mRNA levels, but the effect of homeostasis of FSH on GDNF expression can not be ruled out.2. HMG up-regulated GDNFmRNA and GFRa-1mRNA with increasing dosage of HMG in a dose-dependent manner. As FSH failed to stimulate their mRNA LH contained in HMG may played a major role in up-regulation of GDNFmRNA and GFRa-1mRNA. This suggests that GDNF may be one of the most important regulators which is regulated by LH surge in oocyte maturation and ovulation induction.3. HMG and FSH can not up-regulate expression of RETmRNA by cultured luteinized granulosa cells at the concentration of 5IU/L,10IU/L and 10IU/L respectively. This suggests that other GDNF signal pathways may involved in the process of follicular development and maturation except for GDNF-GFRα-Ret pathway.