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Effects of FSH、LH and Estrodial on the Expression of NPPC Gene in Human Granulose Cell Cells

Author: WangSong
Tutor: QuanSong
School: Southern Medical University,
Course: Obstetrics and Gynaecology
Keywords: granulose cell estradiol progesterone follicle-stimulating hormone luteinizing hormone c-type natriuretic peptides cGMP NPPC oocyte meiosis stasis
CLC: R321.1
Type: Master's thesis
Year: 2013
Downloads: 10
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Abstract


BACKGROUNDNormal meiosis is the key process to ensure the continuity of species which is also the fundamental guarantee of race evolution. It plays an important role in the growth of oocyte and the development of follicular. Meiosis of oocyte is different from the spermatocyte that it is regulated by the initial and terminational signals from the oocyte itself and exogenous. Germ cells go through stages of leptotene, zygotene, pachytene, and finally is blocked in the diplotene stage right before and after birth when germ cells start to form primitive follicles. And the formation of primordial follicle library decides the reproductive ability. In human and mouse ovaries initial meiosis was dated back from embryonic period but was hold in stage of diplotene. Mammals mature oocytes in antral follicle has been stalled in prophase I of meiosis until the LH peak induces the recovery of meiosis and ovulation afterwards. Oocytes which are stagnated in prophase of meiosis can regain their ability of meiosis after generation and accumulation of key molecules in cell cycle regulation that can drive cells resume meiosis process itself. However, despite the oocyte itself, normal meiosis can still be blocked by some factors generated from follicle mesenchymal cells. Premature follicular oocytes of meiosis stagnation before ovulation was to assure mature of oocyte cytoplasm and nucleus with synchronization and the same with oocyte mature and ovulation behavior. In normal reproductive cycle, the hypothalamus secrets LH peak before ovulation to stimulate meiosis of graff follicular oocyte and ovulation. However even without hormone stimulation, follicular oocytes separated and cultured in simple nutritional support condition in vitro was still able to resume meiosis spontaneously. The first obvious morphological hallmark of oocyte meiosis recovery is rupture of germ-vesicle. In1935researchers observed for the first time that germ-vesicle burst spontaneous in vitro culture of rabbit and human oocytes experiment, and put forward the view that the COC compounds was isolated from the rest of the follicle tissue which resulted the separation of meiosis inhibiting factor and complex, thus led to the spontaneous recovery of COCs meiosis. Later similar phenomenon had been observed in other mammal oocytes.Oocyte need a steady rise in cAMP for inhibition of meiosis of GV stage oocytes. PDE3A,a specific phosphodiesterase within oocyte, hydrolyze cAMP in oocyte then activate the growth stimulation proteins and initiate meiosis recovery signal pathway,which led to the subsequent oocyte germ-vesicle burst. Before the LH peak, cGMP generated by cumulus cell are transferred to the oocytes through gap junctions, that inhibited oocyte PDE3A activity thus blocking the declination of cAMP in the oocyte and the GVB occurrence.NPPC is a small polypeptide composed of22amino acids, it expressed in mural granulosa cells surrounding follicle wall in Graafian follicle.And its cogn ate receptor guanosine acyl cyclase NPR2is mainly expressed in cumulus cell surround oocyte. NPPC act on NPR2receptors in cumulus cell to stimulate t hesecration of cGMP. Extract the COCs complex from Graafian follicle and co cul-ture with cells can elevate the amount of cGMP in oocytes and cumulus c ellsand prevent GVB. Studies have shown precocious meiosis of oocytes in NP PC/NPR2disfunction in mice or knockout mice oocytes, which confirmed the im-portant role NPPC and its cognate receptor NPR2in maintaining meiosis in hibited.After Chiyo found NPPC and its cognate receptors the NPR2and believed that this signal system played an important role in the female reproductive system and in maintaining meiosis stagnant state [10], Takehito began study NPPC gene deletion and NPR gene mutant mice ultrastructural morphology of the follicles and oocytes to explore the biological defects caused by the lack of NPPC/NPR in reproductive system. Scientists found in this experiment, the ovulation number of NPR-deficient mice was similar to normal mice, but all the oocytes never eliminated the first polar body.All oocytes discharged by NPR/NPPC deficient mice are biological defected, such as fragmented or TS degradation in the cytoplasm, and can not developed to the2-cell stage even after fertilization. The histological observation of ovarian showed in the NPR2and NPPC deficient mice antral follicles, oocytes meiotic resumption occurred in advance, part of antral follicles oocytes and oocytes collected before ovulation showed the derangement chromosome or messy chromatin [11]. No cumulus cells surrounding oocytes after ovulation and in ovulation follicl of Knock-out mouse were found, these findings show that the NPPC/NPR signal system is essential to oocyte meiotic arrest and cumulus corona formation, and effect female fertility by affecting the developmental potential of oocytes.COCs cultured in vitro in mice confirms that estrogen can stimulate and stabilize the NPPC-22inhibitory function on oocyte meiosis, upregulate and extended to24h. Estrogen empower cumulus cells to produce cGMP under the effect of NPPC in vitro, this allows cumulus cell reactivity on NPPC cultured in vitro is equivalent to those of freshly acquired after ECG stimulation which possess FSH activity [8]. Whatsoever singular FSH stimulation on cumulus cells and granulosa cells cultured In vitro did not increase the NPPC gene and protein expression. Domestic and foreign scholars have confirmed the mouse preovulatory LH surge can reduce ovarian granulosa cells in the CNP content, to reduce combination of CNP and its specific receptor NPRB,and the synthesis of cGMP thus to promote the recovery and mature of preovulatory oocytes. In addition through the pathway of downregulating the expression of NPPC/NPR2system to reduce cGMP cyclic nucleotide, LH may also activate MAPK kinase through the EGF network system to reduce the expression of Cx43, close the gap junction channel connecting granulosa cells and oocytes, thereby reduce cGMP flow spread from cumulus cells metastatic to the oocyte [9] to affect female fertility.In recent years scholars carried out a preliminary study about one of the special behavioral processes in the mammalian oocyte meiotic process meiotic arrest. Based on the research results, they establish an initial meiotic regulation model in mammalian preovulatory follicles as cored of NPPC/NPR2system.The speculative model contains a large membership (FSH, LH, EGF, MAPK, NO, T, E2, ODPFs, NPPC, NPR2, cAMP, cGMP, GPR3/12, ADCY, PDE3A), of which FSH, E2and oocyte paracrine factor ODFs may increase cGMP production through stimulated increasing expression of NPPC and Npr2to maintain meiotic suppression and LH may be downregulate through the the EGF network system or NPPC/NPR2expression to induce cyclic nucleotides reduction, or activate MAPK and thus close the gap junction channel connecting granulosa cells and oocytes, thereby reduce cGMP flow spread from cumulus cells metastatic to the oocyte [9] to affect female fertility.Crosstalk of Different cell types (parietal granulosa cells, cumulus cells, oocytes) and different signaling pathways to each other constitutes complex regulatory networks that maintains oocytes meiosis suppression [6,7,8]. However, the research is still in the preliminary stage of speculation, and the results are controversial.Recently studies have gradually confirmed steroid hormone and gonadotropin has an important role in the regulation of thid core network of the mammalian in vitro. However, it must be noted that previous studies did not study the NPPC gene expression changes style in follicle growth and development of and how different hormones affect its expression in the vivo. Therefore, this study aimed to investigate NPPC gene time-varying style during the the superovulation process along with the development of follicles,to explore realtive factors affecting the expression of the core NPPC/NPR system in vivo, so as to get a further understanding of the maintenance and regulatory underlying mechanism of oocytes meiosis standstill. Thus to provide theoretical basis evidence to solve some clinical oocytes maturation or fertilization disorders in Reproductive medicine.OBJECTIVETo test level of estradiol, progesterone, follicle stimulating hormone, luteinizing hormone,CNP, cGMP, GSH in follicle fluid of different diameter of follicle during superovulation and estradiol, progesterone, follicle stimulating hormone, luteinizing hormone levels,to explore their correlation with of CNP, cGMP in follicle fluid.To compare CNP, cGMP, expression differences of different development stages and before and after injection of HCG. To discuss NPPC gene change in follicle growth and development as well as the possible influence factors y and mechanism in the process of meiosis recovery.METHODSSelected a total of54cases with patients undertook follicular puncture and egg harvest during superovuation in therapy of fertilization/egg cytoplasm within a single sperm injection and embryo transfer (IVF/ICSI-ET) in vitro during September2012-January2013in the south of the reproductive center hospital. Charging different diameter of follicle fluid in the process of piercing and picking up eggs to get a total of80cases, separate granulosa cells. According to the presence of injection of HCG,we divide them into two major groups:injection of HCG group, no injection of HCG group,and further divided into four subgroups according to follicle diameter size. For group A are follicles of diameter from10mm to12mm, group B for follicles of13mm to15mm in diameter, group C of16mm to18mm, group D for diameter>18mm. Using chemiluminescence method for determination of serum estradiol, progesterone and follicle-stimulating hormone, luteinizing hormone concentration, ria method is used to detect concentration of estradiol, progesterone, follicle-stimulating hormone, luteinizing hormone contained in follicle fluid of each group.Applying Enzyme-linked immunosorbent method to test the concentration of cGMP and CNP. Eventually q-PCR is for detection of NPPC gene expression differences and changes in granule cells between different groups.RESULTS(1) Hormone levels within follicle fluid tests showed:estradiol level in different diameter groups have significant difference (P=0.012, P=0.014). Results show that estradiol concentrations within the follicular fluid in HCG injection group or the other group is significantly improved with increased follicle diameter. The comparison of same diameter between the major groups shows that:the estradiol level in the follicular fluid in HCG injection group were lower than no HCG injection group (P values were0.023,0.023,0.017,0.014). Within the HCG injection group and no injection of HCG, progesterone levels of different diameter group shows no significant difference (P=0.24, P=0.282). In the follicles of the same diameter, its progesterone has significant difference between injection and no injection of HCG groups (P=0.025), progesterone levels within the follicular fluid were higher in HCG injection group than no HCG injection group (P values were0.033,0.033,0.031,0.014). FSH levels of different diameter groups had no significant difference (P=0.060). Along with the rising of the follicle growth and follicle diameter, FSH levels within the follicular fluid did not change obviously between groups, in the same follicle diameter, FSH level were no significant difference between groups (P=0.766). LH levels in the group of different diameter had no significant difference. The same diameter of the HCG injection and no injection of HCG group comparison:injection of HCG group within the follicular fluid LH levels were higher than no HCG injection group (P value is0.0000).(2) Granular cell CNP, cGMP detection results show that CNP levels in different diameter of group have significant difference (P=0.047, P=0.042). Diameter groups of CNP concentration gradient within the follicle fluid presents group A>B>C>D, whether it is or is not injected with HCG, with the increase of follicle diameter concentrations of CNP rise significantly. Same diameter of the HCG injection and no injection of HCG group comparison:within the follicular fluid CNP levels were lower in injection group than the other group respectively (P value is0.000). Changes of cGMP level and CNP is consistent in different diameter of groups, whether injection of HCG or not, cGMP concentration rose markedly with the increase of diameter (P=0.042, P=0.042). The same diameter of HCG injection and no injection group comparison:cGMP level is lower in HCG injection group (P values were0.013,0.013,0.035,0.025).Results show that levels of CNP and cGMP level changes are consistent, between groups CNP levels within the follicular fluid and the diameter of follicle and estradiol and FSH concentrations were positively correlated.(3)The results show:NPPC gene expression levels of granulosa cells of different diameter have significant differences. Without injection of HCG,significant differences existed about NPPC gene expression levels of granulosa cells with different diameter groups, mRNA expression upregulated along the increase of follicle diameter (P=0.039), the order from high to low is:group A>B>C> DResults were similar in HCG injection group, the rising level of gene expression occurs (P=0.018). With HCG or not, NPPC gene expression rose with the increased follicle diameter. Within the same diameter group compared36hours after HCG was injected with no injection, NPPC gene expression in granulosa cells (P=0.000) were obviously down-regulated (P=0.000)CONCLUSIONS(1) In the ovarian hyperstimulation process of the corpus luteum long scheme, estradiol concentrations within follicle fluid secreted by granulosa cells in follicular continually increased with the increase of follicle diameter, FSH levels showed a rising trend at the same time. Follicle granulosa cells secreted CNP and cGMP content were also increased along follicular development, the change level are positively related with estradiol and FSH concentrations.(2) NPPCmRNA gene in granulosa cell has already been expressed back from early follicular, and the expression upragulates gradually along with follicular growth, which is consistent with the CNP and cGMP concentration rising trend, and positive correlated with concentration of estradiol, FSH.This indicates that promption of NPPC gene expression during follicular development may be raised by estradiol pathway, and to promote the generation of CNP,Which plays role in synthesis of cGMP in Cumulus cellsto suppress the degradation of cAMP in oocytes.(3)34to36hours after stimulation of HCG, NPPC mRNA gene expression within the follicular granulosa cells appeared downregulated before ovulation, CNP levels were lower before the HCG injection, cGMP synthesis is reduced, and is cohesive with the known high expression of LHR receptor before ovulation of follicular granulosa cells. Therefore, results suggest LH peak before ovulation may cut NPPC by LHR receptor gene expression, reduce synthesis of downstream protein CNP, and inhibiting the production of cGMP from cumulus cells, thus to relieve the inhibition of the degradation of cAMP within the oocytes, and start the oocyte meiosis recovery.

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