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　　每日科学—— 来自马萨诸塞州总医院（MGH）的研究者进行的一项研究表明：因化疗被破坏生育能力的雌性小鼠在接受骨髓移植后，在它们的正常繁殖期内，仍然可以成功妊娠。

　　发表于8月1号《临床肿瘤杂志》上的该研究证实，供体骨髓可通过未知的机制恢复雌性小鼠的生育力。尽管在骨髓移植受体的卵巢中可检测到供体卵细胞或卵母细胞，但受体生产的幼畜均来自受体卵细胞。“这一现象与我们以前的研究结果相符合，来自供体骨髓的细胞进入受体卵巢后将发育成不成熟的卵母细胞。”该研究的主要研究者、MGH文森特繁殖生物中心主任Jonathan Tilly博士说。“尽管目前的研究表明来自供体骨髓细胞的卵母细胞病不能发育成有生育能力的卵子，但移植的骨髓在恢复因化疗而丧失生育能力的雌性小鼠的生育力方面确实发挥了某种作用。

　　在发表于2005年《Cell》杂志上的一篇文章中，Tilly的研究团体发现在接受能破坏生育力剂量的化疗后，接受骨髓移植或血细胞移植的雌性小鼠表现出正常的生育力，并且可在其体内发现未成熟的卵母细胞，这些卵母细胞表达的蛋白标记物表明它们来自于供体细胞。这一研究是2004年Tilly研究团队发表于《自然》上的一篇文章的后续。研究报道说雌性小鼠在成年后仍然能产生卵子，而长期以来人们的观点认为雌性哺乳动物一生只能产生有限数量的卵子，并将随着生命的延续而耗竭，两种观点间存在着差异。所有这些研究报道都极具争议，而眼前的这一研究就是为了延续2005年的研究并解答其他研究者所提出的一些批评意见而设计的。

　　在这一研究中，成年雌性小鼠在接受能致绝育剂量的化疗后一周或两个月接受骨髓移植，移植的骨髓来自于未接受化疗的健康成年雌性小鼠。然后将这些小鼠与健康的成年雄性小鼠共同喂养，并观察7个月，在此期间内，一组对照组的雌性小鼠至少成功的进行了五次妊娠。雄性小鼠和供体雌性小鼠毛色均为黑色，而受体雌性小鼠毛色为白色。这样单凭幼鼠的毛色就可以判断卵细胞的来源：如果幼鼠毛色为棕褐色，卵细胞就来自于受体雌性小鼠，如果为黑色，则卵细胞就来自于骨髓供体。在化疗后一周接受骨髓移植的10只雌性小鼠中，有9只在研究期间均成功妊娠数次。其中一只产下四窝幼鼠，一只产下五窝幼鼠，七只产下六窝幼鼠。所有的幼鼠均为骨髓受体的后代。与之形成对比的是，另外一组13只化疗后未接受骨髓移植的雌性小鼠中，10只有怀孕，但没有一只能产子超过三窝。

　　另外的研究表明化疗后一周骨髓移植的小鼠比化疗后八周接受移植的小鼠的生育能力要好。同样的，在移植后短期内恢复交配也可以提高生育力。当把化疗剂量提高到小鼠的半数致死量后，那些接受骨髓移植的小鼠的生存率和长期生育力均较其他小鼠有所提高。交配试验生产的幼鼠的毛色结果表明骨髓移植虽有助于恢复生育力，但它的作用范围并不包括那些会分化为生殖细胞的细胞。为了进一步探讨这一现象，MGH-Vincent的研究者们将转基因雌性小鼠的骨髓移植给接受化疗的雌性小鼠，这些转基因小鼠的生殖系细胞（即卵母细胞的前体细胞）能表达一种绿荧光蛋白（GFP）标记物。在移植两个月后，研究者们在受体卵巢的未成熟滤泡内检测到GFP标记的卵母细胞。然而，供体来源的卵母细胞仅占滤泡内卵母细胞总数的不足2%，并且成熟滤泡不包含GFP标记的细胞。

　　在那些发表的对Tilly研究团队先前的研究结果持反对意见的论文中（这些论文均未对实验过程加以重复），有人推理说2005年发表于《Cell》上的论文中提到的在受体卵巢中观察到的GFP标记的细胞有可能是供体的免疫细胞，而非卵母细胞。为了反击这一推论，MGH-Vincent的研究队伍分别从正常小鼠、仅生殖系细胞含GFP的小鼠和所有细胞均表达GFP的小鼠体内分离提取免疫细胞。在经过仔细的分析后，他们证实仅生殖系细胞含GFP的小鼠的免疫细胞内并没有蛋白标记物，这说明在接受生殖系细胞被标记过的骨髓移植的雌性小鼠卵巢内发现的GFP标记的细胞只能是卵母细胞。进一步的实验证实提取的免疫细胞并不表达卵母细胞特异性的标记物基因，这些基因先前被Tilly研究团队用来辨别骨髓来源的卵母细胞。

　　Tilly和她的同事解释说，由于大多数保护生育力的物质均需在化疗以前使用才能有效，因而移植的供体骨髓所起的作用可能是恢复而不是保护生育力。“目前，我们并不能确切知道骨髓中的何种物质让受体恢复产生卵母细胞并挽救了受体的长期生育力。但是，我们很确信成熟的骨髓可以产生未成熟的卵母细胞，只是从移植后的结果来看，这些未成熟的卵母细胞对挽救生育力或许并不是非常重要。”Tilly指出，在他们的论文在2005年的《cell》上发表以后，其他研究团队也进行并发表了三项研究，其结果均与他们在雌性小鼠身上得到的结果类似，表明在实验室中和移植入睾丸后，来自成年雄性小鼠或男性的骨髓细胞可被诱导产生未成熟的精子。“很明显，在骨髓中的干细胞有能力分化产生未成熟的卵子和精子这方面尚存在一些未知的东西，我们需要弄清这些东西是什么。”他说。Tilly是哈佛医学院妇产科和生殖生物学的一名副教授。

　　Bone Marrow Restores Fertility In Female Mice, Study Confirms

　　Updated: 7/31/2007 2:07:12 PM

　　Science Daily — A new study from Massachusetts General Hospital (MGH) researchers confirms that female mice that receive bone marrow transplantation after fertility-destroying chemotherapy can go on to have successful pregnancies throughout their normal reproductive life.

　　The report in the August 1 Journal of Clinical Oncology verifies that donor marrow can restore fertility in female mice through an as-yet unidentified mechanism. While donor-derived egg cells or oocytes were observed in the ovaries of marrow recipients, all pups born were from the recipients' own eggs.

　　"Consistent with our past work, cells derived from the donor bone marrow are getting into the ovaries and developing into immature oocytes," says Jonathan Tilly, PhD, director of the Vincent Center for Reproductive Biology at MGH, the study's senior author. "Although these oocytes derived from marrow cells don't appear competent, at least thus far, to make fertilizable eggs, marrow does contribute something that allows a resumption of fertility in female mice sterilized by chemotherapy."

　　In a 2005 paper published in the journal Cell, Tilly's group found that the ovaries of female mice that had received bone marrow or blood cell transplants after fertility-destroying doses of chemotherapy appeared normal and contained immature oocytes expressing a marker protein indicating they came from the donor cells.

　　This report followed a 2004 Nature paper, also from Tilly's team, reporting that female mice continued producing eggs well into adulthood, in contrast to the long-held belief that female mammals are born with a finite supply of eggs that is depleted throughout life. Both those papers have been extremely controversial, and the current study was designed to follow up the 2005 paper and to address criticisms raised by other researchers.

　　In the current study, adult female mice treated with infertility-inducing chemotherapy received bone marrow transplants from non-treated, healthy adult females either one week or two months after chemotherapy. The mice were then housed with healthy adult males and followed for 7 months, a time period in which a group of control females achieved at least five successful pregnancies each.

　　Both the males and the donor females were black in coat color while the recipient females were white-coated. As a result, the coat color of any pups would indicate the source of egg cells used to make the offspring, with tan coats signifying eggs from the recipients and black coats indicating that the eggs had come from marrow donors.

　　Of the 10 females that received bone marrow transplants one week after chemotherapy, all but one achieved several successful pregnancies during the study period. One gave birth to four litters, one gave birth to five litters, and seven gave birth to six litters of pups. All pups were offspring of the recipients. In a comparison group of 13 females that did not receive marrow after chemotherapy, 10 did become pregnant, but none delivered more than three litters.

　　Additional experiments indicated that mice receiving transplants one week after chemotherapy had better fertility outcomes than did those transplanted at eight weeks. Similarly, resuming mating sooner after transplantation also improved fertility rates. When chemotherapy doses were increased to levels expected to cause death in half the mice, those that also received bone marrow transplants had improved rates of both survival and long-term fertility.

　　The coat-color results of the mating trial indicated that the transplanted marrow's contribution to restoring fertility did not involve cells destined to becoming fertilizable eggs. To further investigate this observation, the MGH-Vincent researchers gave chemotherapy-treated females marrow from transgenic females that express a green fluorescent protein (GFP) marker only on germline cells, which are precursor cells involved in producing oocytes. Two months after the transplant, the researchers observed GFP-marked oocytes in immature follicles within recipient ovaries. However, donor-derived oocytes made up less than 2 percent of the total number of oocytes contained within follicles, and no mature follicles contained GFP-marked cells.

　　Among the published reports raising objections to the previous work of Tilly's group -- none of which actually attempted to duplicate those experiments -- one theorized that GFP-marked cells observed in recipient ovaries in the 2005 Cell paper might be donor immune cells rather than oocytes. To address that conjecture, the MGH-Vincent team isolated immune cells from normal mice, from the germline-only GFP strain used in their experiments, and from a strain of mice expressing GFP in all cells.

　　Careful analysis confirmed that no immune cells from the germline-only GFP strain contained the marker protein, making it highly unlikely that GFP-labeled cells in the ovaries of females receiving germline-only-labeled marrow were anything other than oocytes. This was further confirmed by experiments showing that isolated immune cells did not express the oocyte-specific marker genes previously used by Tilly's group to identify the marrow-derived oocytes.

　　Tilly and his colleague note that, since agents that protect fertility most likely would need to be given before chemotherapy to be effective, whatever the donor marrow contributes probably acts by restoring rather than preserving fertility. "Right now, we really don't know exactly what it is in marrow that restores recipient oocyte production and rescues long-term fertility. However, we do know without question that immature oocytes can be generated from cells in adult bone marrow, but they are probably not critical to the fertility rescue observed after the transplants."

　　Since the 2005 Cell paper, Tilly points out, three studies have been published by other groups showing that, similar to his team's work in females, bone marrow cells from adult male mice or from men can be coaxed to make immature sperm cells, both in lab dishes and after transplantation into the testes. "Clearly, something is going on here regarding the ability of stem cells in bone marrow to produce immature egg and sperm cells, and we need to figure out what it is," he says. Tilly is an associate professor of Obstetrics, Gynecology and Reproductive Biology at Harvard Medical School.

　　The first author of the study is Ho-Joon Lee, PhD, of the MGH-Vincent Center for Reproductive Biology. Co-authors are Kaisa Selesniemi, PhD, Yuichi Niikura, PhD, and Teruko Niikura, also of MGH-Vincent; and Rachael Klein and David Dombkowski of the MGH Center for Regenerative Medicine. The work was supported by grants from the National Institutes of Health, Sea Breeze Foundation, JM Foundation and Vincent Memorial Research Funds.

　　Note: This story has been adapted from a news release issued by Massachusetts General Hospital.

　　http://www.dentalplans.com/articles/22297/