摘要
卵巢异性移植对充分利用动物生殖细胞资源具有特殊的应用价值。为探讨卵巢异性移植中两性配子同体发育、受精及胚胎发育能力,本研究通过卵巢异性移植构建雌、雄性腺同体模型,在哺乳动物小鼠中对卵巢异性移植受体雄鼠性腺、精子、相关基因表达进行了检测,并首次对同体发育两性配子体外受精和胚胎发育能力进行了研究,获得以下研究结果:
1.在卵巢移植后21d和28d,卵巢移植体生长发育良好,有大的有腔卵泡发育;卵巢移植受体的睾丸、附睾形态正常,与对照组雄鼠无明显异常,各组间雄鼠体重、睾丸附睾湿重及睾丸附睾与体重的比值均差异不显著(P＞0.05)。组织学检查结果也表明,移植组睾丸生精细管、附睾头和附睾尾的结构正常,管腔内均有精子分布,与对照组比较形态上无明显差异。这表明卵巢移植体未对雄性受体睾丸组织产生明显的影响。
2.卵巢移植体的生长及卵泡发育未对雄性移植受体精子的生成及成熟产生明显影响,雄性受体精子的形态、精子畸形率(7.62%和7.78%)保持在与对照组相近水平,无显著差异。
3.通过卵巢移植受体雄鼠在卵巢移植后21～60 d期间与正常雌鼠合笼配种,观测了卵巢移植体对受体鼠生育能力的影响。结果显示,在卵巢移植后21～60天期间卵巢移植体仍有卵泡发育的生理状态下,试验组受体鼠具有与对照组雄鼠同样的生育能力,试验组的平均窝产仔数(8.83±2.10)与对照组(9.85±2.34)无明显差异。这一结果也与受体鼠精子形态学的观察结果相一致,说明在雌、雄性腺同体的受体鼠中,睾丸精子的发生与成熟过程没有受到卵巢移植体功能活动的明显干扰,受体鼠精子具有体内受精能力并能繁殖正常后代。
4.采用RT-PCR方法对小鼠精子发生及凋亡相关基因Pgk2、Dazl、Prm2和Bax的表达进行了分析,未发现不利于精子发生的表达异常,说明卵巢移植对雄性受体小鼠的精子发生在分子水平上未产生明显的损害作用。
5.通过体外受精试验初次证实,在卵巢异性移植构建的雌、雄性腺同体生理环境中,同体发育的两性配子具有体外受精和胚胎发育至囊胚的潜能。这些同体发育的两性配子可以相互结合完成受精过程,其2-细胞胚胎发育率和囊胚发育率可分别达到60.4%和33.9%。这表明雌雄异体繁殖模式的动物仍保留着雌、雄配子可以同体发育、受精的潜能,同时也说明小鼠雄性个体的生理环境也能满足卵子发育所需的基本条件。
6.本试验通过体外受精和胚胎移植辅助生殖技术,首次成功繁殖了同体发育两性配子的新生后代,共有3只母鼠妊娠并生下6只发育仔鼠,其移植成功率(或妊娠率)、产仔率分别为25.0%和4.2%;观察和比较了试验组与正常繁殖对照组新生仔鼠3周哺乳期的体重增长变化,结果表明两组新生仔鼠3周龄体重及增长趋势基本相同。结果表明,在卵巢异性移植受体中,雌、雄配子不仅具有同体发育、受精潜能,而且可以通过体外受精和胚胎移植辅助生殖技术繁殖新生后代。其新生仔鼠具有正常生长发育能力。
7.研究对雌雄性腺同体发育两性配子体外受精繁殖的后代雄鼠进行了生育能力评价。试验组2只后代雄鼠与正常雌鼠配种繁殖,均能使母鼠受孕,平均窝产仔数达9.60±3.98只,与正常雌、雄鼠配种对照组相比无显著差异(P＞0.05)。结果表明卵巢异体移植后同体发育配子产生的新生后代具有正常繁殖能力,配子同体发育不影响其子代的繁殖能力。
Ovarian heterosexual transplantation has special value for full utilization of germ cells as a resource in female animals. In order to evaluate the competence of fertilization and embryo development on the female and male gametes developed in the male recipients, ovarian heterosexual transplantation were used to produce the mouse model with both female and male gonads for examining sexual glands of male recipients, sperms and related gene expression. For the first time in mammals, the competence of the female and male gametes produced in the male graft recipients was assessed by performing in vitro fertilization and embryo transfer in this study. The main results were following:
1. Ovarian grafts developed well with large antral follicles on day 21 or 28 after transplantation. Morphology of testis and epididymis from the ovary graft recipients was normal, without significant difference compared with that control male mice. The body weight, wet weight of testis and epididymis, and the weight ratio to the body weight of male mice in each group had no statistics difference (P>0.05). It was shown that The seminiferous tubule in testis, epididymis from the ovary graft recipient were normal structure and the lumen of the tubes were filled with sperm by histological section observation, appeared no difference in morphology compared with that control males. The present study demonstrates that ovarian allograft do not appear to greatly affect testis, epididymis structure from the ovary graft recipient
2. The sperm abnormal rate in 21 d and 28 d ovarian translantation group was 7.62% and 7.78%, respectively ,without significant difference compared with that control group(6.73%) (P>0.05). The growen ovarian grafts and developed follicles do not appear to significantly affect spermatogenesis in testis and sperm maturation in epididymis of male recipients.
3. Reproductive capacity of the ovary graft recipients with developed follicles in ovarian grafts was investigated by breed with normal female mice during 21-60 d after ovarian tansplantation. The litter size in the experimental group was 8.83±2.10, without significant difference compared with that control group(9.85±2.34) (P>0.05). It demonstrates that the ovary graft recipients are fertile, the functional ovarian grafts do not appear to significantly affect spermatogenesis, sperm maturation, and fertilizing ability in vivo of the male recipients.
4. Expression of Dazl, Pgk2, Prm2 and Bax mRNA in testis and epididymis were determined by using reverse transcriptase-polymerase chain reaction. The expression of 4 gene mRNAs in 2 experimental groups showed similar trend with the the control group. It indicates that the ovarian transplantation do not appear to significantly affect spermatogenesis in the ovary graft recipients at the molecular level.
5. Oocytes and sperms collected from the same ovary graft recipient can fertilize in vitro. The two-cell cleavage rate and blastocyst rate in the experimental group was 60.4% and 33.9% , respectively. This is the first report that both sexual gametes developed in same physical environment in mice with male and female sex glands by heterosexual ovarian transplantation present potential of fertilization in vitro and development to blastocyst. This work has shown that the physical environment of male mice can support the development of oocytes in ovarian allografts and sexual reproduction in animal preserves potential of male and female gametes developing in same body.
6. The developmental competence of oocytes and sperms collected from the same ovary graft recipient was assessed by performing in vitro fertilisation and embryo transfer to recipients. Six pups developed normally were born by three surrogate mothers. The pregnancy rate and total birth rate was 25.0% and 4.2%, respectively. This is the first report of live offspring obtained from both sexual gametes developed in same body after heterosexual ovarian transplantation using IVF. The growth of the pups in the experimental group and control group was evaluated by measuring the body weight on day 1, 7, 10, 14 and 20 after birth. The growth of the pups showed similar trend between the experimental group and control group. In conclusion, this work has shown that male and female gametes from recipient mice with male and female sex glands established by heterosexual ovarian transplantation present potential of development and fertilization and that these gametes can produce live offspring. The offspring can develop normally.
7. The reproductive competence of offspring produced by in vitro fertilization with both sexual gametes from ovarian grafts and testes of male recipients was examined. An average litter size up to 9.60±3.98 in the normal female mice mated with 2 male offspring was observed in test group and showed no significant difference with control group. The results demonstrate that the offspring produced by both sexual gametes from ovarian grafts and testes of male recipients have normal reproductive capacity, without impact on it.
引文
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