HORMONE CONTROL OF SPERMATOGONIAL ARREST IN MUTANT MICE

幼年精原细胞耗竭（jsd）小鼠在青春期开始精子发生的浪潮，但随后精子分化停止，尽管继续存在的A精原细胞。 虽然其机制尚不清楚，但我们发现睾酮（T）抑制精原细胞分化，而GnRH拮抗剂或雌二醇（E2）可以恢复精原细胞分化。 我们推测，抑制精原细胞分化的jsd小鼠是由于T的作用，在一个特定的体细胞（Sertoli，Leydig，管周肌样，或小动脉平滑肌）的基因表达。 我们利用小鼠遗传学的力量来阐明T介导jsd小鼠精原细胞“停滞”的机制，并开始鉴定涉及以下目的的细胞类型和生殖调控基因：（I）为了确定雄激素而不是FSH的特异性作用，我们将使用也携带雄激素受体（AR）和FSH β基因突变的jsd小鼠完成研究。 为了确定E2的作用模式和作用部位，将在jsd小鼠中比较GnRH拮抗剂和E2治疗的精子发生恢复与携带雌激素受体（ER）-α或ER β突变的jsd小鼠中的精子发生恢复。 (II)为了证实激素通过直接作用于睾丸和/或曲细精管来影响精原细胞分化，我们将在jsd小鼠睾丸组织和曲细精管的体外培养中检测它们对精原细胞的影响。 (III)为了鉴定精原细胞分化的激素抑制靶细胞，我们将在jsd小鼠和AR缺陷型jsd小鼠之间移植小管，并使用由不同体细胞特异性启动子驱动的Cre重组酶，使用具有loxP位点的AR基因的细胞类型特异性消除。(IV)为了确定相关基因，我们将利用基因芯片在jsd小鼠的靶细胞中差异筛选候选的转录调控基因。 一个好的候选基因必须在GnRH拮抗剂的作用下向一个方向改变其水平，而在T的作用下向相反的方向改变其水平。对jsd小鼠精原细胞“停滞”及其逆转机制的阐明可以应用于遗传决定的雄性不育病例。 由于这一结果与毒物处理和衰老大鼠精原细胞分化障碍的相似性，因此也适用于生殖毒物引起的无精子症和衰老引起的精子发生功能下降。

Juvenile spermatogonial depletion (jsd) mice initiate a wave of spermatogenesis at puberty, but then sperm differentiation ceases despite the continued presence of A spermatogonia. Although the mechanism is not known, we showed that testosterone (T) inhibits spermatogonial differentiation, which can be restored with GnRH antagonist or estradiol (E2). We hypothesize that inhibition of spermatogonial differentiation in jsd mice is due to the action of T on gene expression in a specific somatic cell (Sertoli, Leydig, peritubular myoid, or arteriolar smooth muscle). We utilize the power of mouse genetics to elucidate the mechanism by which T mediates spermatogonial "arrest" in jsd mice and begin identifying the cell type and hormonally regulated gene(s) involved in the following Aims: (I) To establish the specific role of androgen and not FSH, we will complete studies using jsd mice also carrying mutations in the androgen receptor (AR) and FSHbeta genes. To determine the mode and the site of action of E2, the restoration of spermatogenesis with GnRH-antagonist and E2 treatment will be compared in jsd mice with that in jsd mice carrying mutations for estrogen receptor (ER)-alpha or for ERbeta. (II) To establish that the hormones affect spermatogonial differentiation by direct action on the testis and/or seminiferous tubules, we will examine their effects on spermatogonia in in vitro cultures of testicular tissue and tubules from jsd mice. (III) To identify the cell that is the target for hormonal inhibition of spermatogonial differentiation, we will transplant tubules between jsd mice and AR-deficient jsd mice and use cell-type specific elimination of an AR gene with loxP sites using Cre-recombinase driven by promoters specific for the different somatic cells. (IV) To identify the responsible gene, we will differentially screen for candidate hormone-regulated genes in the target cell of jsd mice using microarrays. A good candidate gene must have its level changed by GnRH antagonist in one direction and by T in the opposite direction. Elucidation of the mechanism of this spermatogonial "arrest" and its reversal in the jsd mouse could apply to cases of genetically determined male infertility. Because of its remarkable similarity to the failure of spermatogonial differentiation in toxicant-treated and aging rats, these results could also apply to azoospermia induced by reproductive toxicants and the decline in spermatogenesis with aging.