小鼠卵巢冷冻移植后卵泡发育和卵母细胞成熟的研究

Investigation of follicular development and oocyte maturation after cryopreservation and xenograft of newborn mouse ovaries

本研究探讨了冷冻保存的1日龄小鼠卵巢异体异位移植后,其原始卵泡重新启动生长发育的能力。一日龄B6C2F1 小鼠卵巢分离冷冻后置液氮中保存,保存1周-6个月后解冻,并将卵巢移植到8-12周龄B6C2F1受体鼠肾脏包膜下,移植至少14 d。每侧肾囊移植2枚卵巢的40只受体鼠中卵巢的回收率为45．00％(72/160),而每侧肾囊移植1枚卵巢的20只受体鼠的回收率为82．50％(33/40)。移植卵巢上卵泡的发育基本与体外自然生长鼠的卵巢卵泡发育情况一致。对卵巢移植 19 d的受体鼠用孕马血清促性腺激素(pregnant mare serum gonadotrophin,PMSG)处理后,从移植卵巢上发育成熟卵泡中获得的卵母细胞在MEMα培养基中培养16-17 h,有40．90％,的卵母细胞发生生发泡破裂(germinal vesicle breakdown,GVBD), 其中89．02％的卵母细胞发育到第二次减数分裂中期(metaphase Ⅱ,MⅡ)。将剩余的卵母细胞继续培养到20-21 h,又有50．83％的卵母细胞发生生发泡破裂,但其中只有21．40％的卵母细胞能够发育到MⅡ期。以上结果说明,小鼠早期卵巢经过冷冻- 解冻并异体异位移植后,其原始卵泡能够重新启动生长发育,发育后的卵泡卵母细胞能够在体外培养成熟。这些结果意味着原始卵泡或卵巢冷冻-移植技术有可能充分利用雌性生殖细胞用于濒危动物保种、建立动物基因库和人类辅助生殖等。

In order to explore the feasibility of cryopreserving primordial follicles in attaining their developmental competence following freezing and thawing, ovaries from newborn mice were cryopreserved and the thawed ovaries were xenografted into kidney capsules of adult female mice. Ovaries were isolated from newborn B6C2F1 female mice, infiltrated by Leibovitz 15 （L-15） medium containing 10% （V/V） fetal bovine serum （FBS） and 1.5 mol/L dimethylsulfoxide （DMSO）, and then packed into 0.25 ml plastic straws. The ovaries contained in straws were frozen under nitrogen vapour at -40℃ in Cryocen 1200 programmable freezer, and stored in liquid nitrogen for periods ranging from 1 week to 6 months. Upon thawing, the straws were dipped into room temperature water for 10～20 s, after which the ovaries were collected and washed in L-15 buffer containing 10% （V/V） FBS without DMSO to remove cryoprotectant. The thawed ovaries were transplanted into kidney capsules of 8~12-week old adult B6C2F1 female recipient mice by two protocols, with either 1 or 2 ovaries in each capsule. Upon withdrawal after at least 14 d of transplantation, only 45.00% （72/160） of the ovaries were recovered from 40 recipients transplanted with 2 ovaries in each capsule, compared to 82.50% （33/40） in 20 recipients with only 1 ovary in each capsule. The grafted ovaries exhibited similar follicular developmental progression to that of natural ovaries. There were antral follicles present in the transplanted ovaries on day 14, whose number increased more substantially on day 19 after transplantation. Following stimulation of the recipient mice with 10 IU PMSG on day 19 after xenografting, follicles further developed to preovulatory stage with appearance of cumulus oocytes and enlarged antrum. Oocytes from these fully grown antral follicles were collected andmatured in vitro in modified essential medium-α （MEMt~）. After 16～17 h of culture, 40.90% of the oocytes exhibited germinal vesicle breakdown （GVBD） and among which 89.02% proceeded to the metaphase Ⅱ （MⅡ） stage as indicated by exclusion of the first polar body. The remaining oocytes were further cultured and 50.83% of which initiated GVBD by 20～21 h of culture, but only 21.40% of which proceeded to MII. The above results demonstrated that the primordial follicles in newborn mouse ovaries were capable of sustaining freezing and thawing, and reinitiating development following xenograft into kidney capsule in adult recipient female mice. Production of mature oocytes from such re-developed follicles following gonadotrophin priming and the subsequent oocyte in vitro maturation implied immense prospect of application of this method to preserve female germ cells, conserve endangered species, establish animal gene stock, and utilize oocytes in assisted reproductive techniques.