Fertility in the Klinefelter syndrome by testicular stem cell transplantation - an experimantal approach in the XXY mice

人类男性不育症最常见的遗传原因是Klinefelter综合征（KS，47，XXY），其导致睾丸功能衰竭、生殖细胞丢失和不同程度的雄激素缺乏（由促性腺激素变化引起）。虽然KS是众所周知的，但尚不清楚导致仅支持细胞（SCO）综合征的生殖细胞丢失是由于生殖细胞系缺陷，睾丸环境紊乱还是两者兼而有之。许多Klinefelter患者参加我们的不孕症研究所。任何可能的治疗方法来克服这种不孕症需要从实验工作的结果。拟议的项目旨在阐明Klinefelter患者获得生育能力的治疗方法的先决条件，例如通过利用自体干细胞。由于在这个阶段，这样的工作不能在患者自身中进行，因此产生和使用适当的动物模型是不可缺少的。带有额外X染色体（核型41，XXY）的小鼠模仿患者中发现的条件，通过饲养带有自发突变Y染色体的动物产生。建立了B6 Ei.Lt-Y系小鼠群体，获得41只（XXY）雄性小鼠，用荧光原位杂交（FISH）技术对活体动物进行了核型分析。我们将显示是否睾丸环境和垂体性腺轴的男性轴承额外的X染色体能够支持正常的，二倍体精原干细胞的发展，使用我们成熟的生殖细胞移植和睾丸组织移植的方法。我们将描述男性内分泌学和睾丸功能的重要方面，特别是Leydig细胞的功能和成熟。由于一些Klinefelter患者产生少量的精子来自于明显正常的二倍体精原细胞，睾丸环境应该能够支持精子发生。然而，这是否可能在每个患者或取决于镶嵌的程度还没有得到澄清。染色体异常率高的睾丸精子会增加流产率。因此，在提供任何临床移植方法之前，必须解决睾丸环境紊乱和非整倍体风险增加的问题。这个问题将在小鼠模型中通过检查生殖细胞移植后产生的精子和进行ICSI，然后进行胚胎染色体分析来解决。

The most common genetic cause of human male infertility is the Klinefelter syndrome (KS, 47,XXY) resulting in testicular failure, loss of germ cells and variable degrees of androgen deficiency paralleled by changes in gonadotropins. Although the KS is well known, it is not clear whether the loss of germ cells resulting in a Sertoli-cell-only (SCO) syndrome is due to a defect of the germ cell line, a disturbance of the testicular environment, or a combination of both. Many Klinefelter patients attend our Institute for infertility. Any possible therapeutic approaches to overcome this infertility requires findings from experimental work. The proposed project seeks to elucidate those prerequisites for therapeutic approaches to gain fertility in Klinefelter patients, e.g. by making use of autologous stem cells. Since at this stage such work cannot be performed in the patients themselves, the generation and use of an appropriate animal model is indespensable. Mice bearing a supernumerary X-chromosome (karyotype 41, XXY) mimicking the conditions found in patients have been generated by breeding animals with a spontaneously mutated Y-chromosome. We established a mouse colony of the strain B6Ei.Lt-Y to obtain 41 (XXY) male mice and determined the distinct karyotype of the living animals individually by fluorescence in situ hybridisation (FISH). We will show whether the testicular environment and the pituitary-gonadal axis in males bearing a supernumerary X-chromosome are able to support development of normal, diploid spermatogonial stem cells using our well-established methods of germ cell transplantation and grafting of testicular tissue. We will characterize aspects essential for male endocrinology and testicular function as in particular the Leydig cell function and maturation. Since some Klinefelter patients produce a small amount of sperm deriving from apparently normal diploid spermatogonia, the testicular environment should be able to support spermatogenesis. However, whether this would be possible in every patient or depends on the degree of mosaicism has not been clarified. Testicular sperm with a high rate of chromosomal abnormalities increase the abortion rate. Thus questions of disturbed testicular environment and an increased risk of aneuploidy have to be solved before any clinical transplantation approach can be offered. This problem will be addressed in the mouse model by examining sperm produced after germ cell transplantation and by performing ICSI followed by chromosomal analysis of embryos.