【作者】 袁安文；

【导师】 薛立群；

【作者基本信息】 湖南农业大学 ， 动物遗传育种与繁殖， 2007， 博士

【摘要】 卵巢移植技术对雌性动物生殖细胞资源的保存和利用具有潜在应用价值。移植卵巢不仅可以在雌性移植受体中存活发育,而且成年期卵巢也可在雄性移植受体中存活并维持卵泡发育。为了进一步探索小鼠异性异位移植卵巢卵泡及卵母细胞生长发育规律,本研究将1日龄小鼠卵巢移植到雄性受体肾囊下,把移植受体鼠分为睾丸在位受体组、切除睾丸受体组和促性腺激素处理受体组,并以同期雌鼠为对照,于移植后21～49d回收卵巢移植体,采用形态学、组织学、体外成熟、体外受精和RT-PCR等方法对卵泡发育、卵母细胞生长及卵泡发育相关基因的表达进行研究。结果如下:在264只移植雄性受体中共移植1日龄小鼠卵巢588个,受体总有效率为84.5%,卵巢总回收率为73.3%;移植21d、28d、35d、42d和49d后,移植卵巢显著生长增大(P<0.01),其生长趋势与正常雌性小鼠在位卵巢基本一致;外源性促性腺激素处理能够显著增加移植卵巢平均直径(P<0.05)。初生期移植卵巢能够在雄性在位睾丸受体内完成原始卵泡的形成、募集和生长发育;移植后21～49d回收的卵巢移植体中有各级生长卵泡发育并能达到成熟卵泡阶段,未观察到黄体;切除受体睾丸对移植卵巢卵泡生长发育无明显影响,但在49d卵巢移植体可观察到黄体;外源性促性腺激素处理使卵泡发生排卵前变化,卵丘细胞扩展、黏液化,形成黄体。从在位睾丸受体回收有腔卵泡的卵母细胞主要为GV期,28d卵巢移植体可分离到MⅡ期卵母细胞;从21d、28d和35d卵巢移植体中回收有腔卵泡的卵母细胞数量分别为40.5±29、28.4±21.1和11.1±7.6个,35d卵巢移植体的卵母细胞数量显著低于21d移植体(P<0.05);卵母细胞的平均直径分别为73.6±2.5、74.6±3.4和74.7±5.3μm,35d卵巢移植体卵母细胞平均直径显著大于21d移植体(P<0.05);试验期内,随着移植时间的延长,回收卵母细胞的数量呈减少的趋势,卵母细胞的平均直径有增大的趋势。在切除睾丸受体中,从21d卵巢移植体回收的卵母细胞主要为GV期卵母细胞,有的发育到MⅡ期;回收的卵母细胞数量为32.8±14.8个,平均直径为73.6±3.6μm,均与同期睾丸在位受体组差异不显著(P>0.05)。外源性促性腺激素处理雄性受体21和28d卵巢移植体卵母细胞产量分别为31.5±20.3和28.9±21.2个,与同期睾丸在位受体卵母细胞产量差异不显著(P>0.05),成熟卵母细胞产量分别为9.0±7.9和14.6±13.4个,显著高于同期睾丸在位受体(P<0.01)。外源性促性腺激素不能增加卵母细胞产量,但能增加成熟卵母细胞产量。睾丸在位受体组21d卵巢移植体GV期卵母细胞经IVM、IVF,2-细胞胚胎发育率为16.0%,能够发育到桑葚胚,未发育到囊胚。外源性促性腺激素处理组21d卵巢移植体GV期卵母细胞2-细胞胚胎发育率为32.8%、囊胚发育率为1.3%,极显著高于睾丸在位受体组(P<0.01);MⅡ期卵母细胞2-细胞胚胎发育率37.6%,显著高于GV期卵母细胞(P<0.05),囊胚发育率为12.8%,显著高于GV期卵母细胞(P<0.01)。异性异位移植卵巢卵母细胞能在体外完成正常的受精过程,外源性促性腺激素能够促进卵母细胞的发育能力。移植卵巢和正常卵巢均可检测到卵泡生长、分化和闭锁相关基因GDF-9、AMH、FSHR、LHR、ERβ、P450-arom、P450-scc、IGFBP 4、Bax和Bcl-2 mRNA的表达。卵巢移植、受体的性别及其性腺对目的基因mRNA的表达无显著影响;外源性促性腺激素促进LHR的表达。表明小鼠移植卵巢卵泡生长、分化和闭锁的方式可能与正常卵巢一样,小鼠异性异位移植卵巢卵泡具有正常生长发育和成熟的能力。更多还原

【Abstract】 Ovarian transplantation is an emerging technology with potential value applied to preservation and utilization of female gametes. Ovaries grafted can survive and develop in female recipients, and mature ovaries grafted can survive and develop in male recipients.To investigate further the development of oocytes and follicles of ovarian allografts in male recipient mice,ovaries from one-day-old mice were allografted underneath the kidney capsule of male recipients. The recipients were treated with or without castration and/or gonadotrophins, and same age female ovaries as control of ovarian grafts.Ovarian grafts were recovered 21-49 days after transplantation.Development of the oocytes and follicles of ovarian grafts and expression of genes related to follicular development were evaluated with morphology, histology,in vitro maturation, in vitro fertilization and semiquantitative RT-PCR. The results are as follows.Two hundred and sixty-four male recipient mice were used and 588 ovaries from one-day-old mice grafted. Ovarian grafts were successfully recovered from 84.5 % of the male recipients in total, and the ovary recovery rate was 73.3% in total. Ovarian grafts grew and increased in size significantly (P<0.01) 21, 28, 35, 42 and 49 days after transplantation, and the mean diameter of ovarian grafts increased significantly(P <0.05) after stimulated with exogenous gonadotrophins. Ovarian grafts displayed a pattern of growth similar to that observed in in situ mouse ovaries.Primordial follicle formation, recruitment and growth can be initiated in ovarian grafts in male recipient mice. Ovarian grafts contained follicles at all stages of folliculogenesis and the follicles can develop to maturation stage 21-49 days after transplantation, but didn’t find corpus luteum in ovarian grafts. Ovarian grafts transplanted under the kidney capsule of castrated male mice displayed a pattern of development of follicles similar to that observed in intact male mice, but with corpus luteum formation in ovarian graft 49 days after transplantation. The follicles could develop to the antral stage and undergo periovulatory changes following xenografting and exposure to a luteinizing stimulus, with expanded cumulus-oocyte complex, mucified cumulus cell and corpus luteum.The majority of oocytes collected from the grafts in intact male recipients were at the GV stage. Mature metaphase II oocytes as well as immature GV oocytes were retrieved from the 28 d grafts. The yield of oocytes per ovarian graft in the 21 d, 28 d and 35 d groups was 40.5±29, 28.4±21.1 and 11.1±7.6, respectively. Ovarian grafts in the 35 d group had a significantly lower oocyte yield than that in the 21 d group(P<0.05). The mean diameter of oocytes from ovarian graft in the 21 d, 28 d and 35 d groups was 73.6±2.5, 74.6±3.4 and 74.7±5.3μm, respectively, with significant difference in the mean diameter of oocytes between the 21 d group and 35 d group (P<0.05). It seems that the yield of oocytes per ovarian graft decreases and the mean diameter of oocytes from ovarian graft increases with the graft period extended during test period.Mature metaphase II oocytes as well as immature GV oocytes were retrieved from the 21 d grafts in castrated male recipients. The yield of oocytes per ovarian graft was 32.8±14.8, and the mean diameter of oocytes was 73.6±3.6μm, without significant difference compared with that in intact male recipients(P>0.05).The yield of oocytes per ovarian graft in intact male recipients in the 21 d, and 28 d gonadotrophin-treated groups was 31.5±20.3 and 28.9±21.2, respectively, without significant difference compared with the non-treated groups (P>0.05). The yield of mature metaphase II oocytes was 9.0±7.9 and 14.6±13.4, respectively, with significant difference compared with the non-treated groups(P <0.01). Stimulation with exogenous gonadotrophins did not significantly increase oocyte yield from grafted ovaries but did enhance oocyte maturation.The two-cell cleavage rate of in vitro matured GV oocytes collected from non-stimulated 21 d grafts was 16.0%.Some of the two-cell embryos cleaved to morula but not to blastocyst. GV oocytes from 21 d grafts of gonadotrophin treated recipients had a higher two-cell cleavage rate and blastocyst rate after IVM and IVF (32.8% and 1.3%, respectively) than the oocytes from non-stimulated 21 d grafts(P <0.01). There was significant difference in the cleavage rate (37.6%)( P<0.05) and blastocyst rate (12.8%)( P<0.07)between GV and MII eggs from from 21 d grafts of gonadotrophin treated recipients. Oocytes collected from ovarian grafts in intact male recipients can be fertilized in vitro, and stimulation with exogenous gonadotrophins can improve oocyte development.GDF-9, AMH, FSHR, LHR, ERβ, P450-arom, P450-scc, IGFBP 4, Bax and Bcl-2 mRNA expression were detected in ovarian grafts and normal ovaries. Expression of target genes related to follicular development, differentiation and follicle atresia was not influenced by transplantation, sex and glands of recipients. Exogenous gonadotrophins stimulation upregulated the expression of LHR. In conclusion, the pattern of follicular development, differentiation and follicle atresia in ovarian grafts may be similar to the ovaries of normal female mice. The follicles in ovarian grafts from intact male recipients can grow and develop to maturation stage.更多还原