Study On Cryopreservation And Autotransplantation Of Rat Ovarian Tissue

Cryopreserved ovarian tissue transplantation technique is thattransplanting the pieces of the ovarian cortex to the recipientsandinducing the follicles develop. Althouth the cryopreservation hasprogression, it is still in the research stage, and it cann't used inclinic extensively. How to elevate the developmental potency, decreacethe incidence of apoptosis, and promote the ovarian angiogenesisproduction shoud be solved. There are three parts in this research, whichstudy primariely the effect of control slow cooling protocol on theprimordial and primary follicles, the action of gonadotropin FSH on theexpression of VEGFm RNA and follicular development in cultured andimplantational rat ovarian tissue.Part 1 The effect of contral slow cooling protocol on the primordialand primary follicles and VEGFm RNA expressionObjective To study the effect of cryopreservation on the primordial andprimary follicles and VEGFm RNA expression observation the morphologyand apoptosis of the primordial and primary follicles.Method The immature female SD rats aged 4-6 weeks are used in the study.The ovaries were cryopreserved by control slow cooling protocol. Theovaries were divided into fore groups: fresh group, cryopreserved group,fresh cultured group andcryopreserved cultured group, the Themorphology and apoptosis of the primordial and primary follicles wereobserved. And the VEGFm RNA expression were determined by RT-PCR.Result (1) Compared with fresh grouop, the abnormal morph rate ofprimordial in cryopreserved group has no different (P＞0.05), the ratof abnormal morph of primary, second and antral follicle were lower thanfresh group (P＜0.01). Compared with fresh cultured group,, therat of abnormal morph of primordial and primary follicle in the cryopreserved cultured group were lower (P＜0.01). (2) Both of the ratof injured oocyte and granulose in primary follicle werehigher than fresh group. After in vitro culture, both of therat of injured oocyte and injured granulose in primordial andprimary follicle after cryopreservation increasedsignificantly(P＜0.01). (3) Threr were no differents in apoptosisrate of primordial and primary follicle betweenfresh group and cryopreserved group. After in vitro culture, theapoptosis rate of primordial and primaryfollicle, as well asoocytes and granulose cells were increased significantly(P＜0.01). (4) VEGF mRNA expression decreased aftercryopreservation.Conclusion (1) Cryopreservation may make damage to the primordial andgrowth follicle. With the development of the follicle growth, the degreeof the damage increase. (2) Cryopreservation may induce follicle apoptosis.Apoptosis may be one of the mechanisms of the freezing injure. (3)Cryopreservation may decrease VEGF mRNA expression of ovariantissue.Part2 The effectiveness of FSH on apoptosis of cultured ratgranulose cells and VEGF mRNA expression of ovariantissue in vitroAfter cryopreservation, the oocytes were damaged, as wellas granulose cells, and the VEGF mRNA expression of ovariantissue Decreased. One of the important thongs need to beresolved is that, to elevate the developmental potency of thecryopreserved granulose cells, decrease the apoptosis rat andpromote the Angiogenesis. In this part, we will study theeffectiveness of FSH on apoptosis of cultured rat granulose cells and VEGF mRNA expression of ovarian tissue in vitro byadding FSH in the culture medium.Method the granulose cells were isolated from fresh andcryopreserved ovarian tissue respectively, cultured for 4 days.Thegranulose cells apoptosis rate and VEGF mRNA expression of theovarian tissuein in culture medium with FSH were comparedwith the medium without FSH.Result (1) The apoptosis rate of cryopreserved granulose cells decreasedin the medium with FSH. (2) The granulose cells secreted less VEGF afterfreezing. Both the fresh and the cryopreserved granulose cells secretedVEGF increase with the medium adding FSH. (3) VEGF mRNA expression ofthe ovarian tissue increased with the FSH medium.Conclusion The apoptosis rate of the granulose cells isolatedfrom fresh and cryopreserved follicles decreased by adding FSH in themedium. (2) The level of VEGF secreted by the granulose cells isolatedfrom fresh and cryopreserved follicles increased by adding FSH in themedium, so did the VEGF mRNA expression.Part 3 The empirical study of the cryopreserved rat ovarian tissueautograftingTo study the effectiveness of FSH on the VEGF mRNA expression, thesurvival and development of the follicles by setting up rat ovarian tissueautografting.Method The cryopreserved ovarian tissue autotransplantedinto the granulation tissue in the muscle prefabricated previously. Onegroup administrated FSH once a day, the other group administrated saline.The number and the rate of the follicles, the expression of the VEGF mRNAand PCNA were observed.Result (1)The number of the follicles decreased significantly after transplantation. The number of primordial and primary follicles in FSHgroup increased compared with saline group. (2) The primordial folliclepercentage decreased, and the percentage of the primary and secondaryfollicle increased. The percentage of the primordial follicle decreasedand the primary had increased tendency in FSH group compared with thesaline group. (3) The expression of VEGFmRNA of the two groups elevated48 hours after transplantation., but there was no differents between thetwo groups. The expression of VEGFmRNA in saline group decreased,andmaintain the high level in the FSH group after 10 days. (4) The expressionof PCNA in FSH group was higher than in saline group.Conclusion The exogenous FSH may increase the expression ofVEGFmRNA in autografts, which may decrease the ischemic -reperfuseinjury. (2) The exogenous FSH may promote the survival, development andsecretion of the follicles.