GENE EXPRESSION IN SPERMATOGENIC CELLS

工作概述：长期目标是确定和描述控制男性生殖细胞发育或功能的关键机制。目前使用的方法是鉴定在男性生殖细胞中特异性表达的基因，使用转基因小鼠和体外试验鉴定调节其表达的启动子元件和转录因子，并应用基因敲除方法确定它们编码的蛋白质的作用。许多基因只在男性生殖细胞中表达，我们关注的是少数可能对配子发育或功能很重要的基因。我们分离了糖酵解途径中关键同工酶甘油醛3-磷酸脱氢酶（GAPD-S）的小鼠和人类基因，该酶仅在男性生殖细胞中表达，被认为在调节受精所需ATP的产生中起关键作用。合成肽制备的抗体将GAPD-S定位在小鼠和人精子鞭毛纤维鞘上。酵母双杂交筛选鉴定了一个ww结构域蛋白（FBP11），该蛋白与小鼠GAPD-S的脯氨酸丰富区域结合。我们正在绘制每个蛋白上的结合域，并测试FBP11将GAPD-S锚定在纤维鞘上的假设。此外，分子模拟研究表明，底物结合袋周围的残基可能解释了抑制剂对体细胞和生殖细胞同工酶的影响差异（合作者Bienstock）。人类重组GAPD-S正在被表达用于核磁共振研究，以检查GAPD-S与其天然底物、天然辅因子和生殖毒物的相互作用（合作者伦敦）。其中之一是(S)-3-氯醛，它是工业溶剂环氧氯丙烷的代谢物，似乎是GAPD-S结合底物的竞争性抑制剂。此外，一种Gapds基因靶向构建正在准备中，以验证一种假设，即来自基因敲除小鼠的精子将无法产生达到过度激活运动所需的ATP，也无法使卵子受精（合作者Bunch和O?Brien）。我们之前证明了纤维鞘成分1 （FSC1）是精子鞭毛中的主要细胞骨架蛋白，GAPD-S和FSC1在小鼠精子的这一区域共定位。为了研究它们之间可能的相互作用以及FSC1的作用，我们分离了FSC1 cDNA和基因，并用酵母双杂交法鉴定了相关蛋白。FSC1被发现是一种蛋白激酶a （PKA）锚定蛋白（AKAP），这表明PKA锚定在纤维鞘上，参与了激活精子运动所必需的蛋白proprohorylation事件。酵母双杂交实验、丙氨酸和缬氨酸扫描诱变和拉下实验确定了与PKA四聚体不同调控亚基结合特异性相关的氨基酸。在FSC1中发现的RI&#61474；-特异性和双RI&#61474；/RII&#61474；-特异性结合基序首次在AKAP中进行了表征。研究发现，PKA结合需要akap上三个一致位置的疏水氨基酸，而PKA结合的特异性取决于中间位置氨基酸上脂肪侧链的大小。通过在这些基序中引入点突变来在RI&#61474；特异性、RII&#61474；特异性和RI&#61474；/RII&#61474；双特异性结合之间切换，证实了这一点。这些发现在理解一级序列与决定PKA结合特异性的AKAP锚定结构域的两亲性&#61474；-螺旋内残基的三维空间分布之间的关系方面取得了重要进展。它们对于促进对PKA亚型在细胞内定位的分子机制的理解也具有重要意义。其他研究正在使用酵母双杂交筛选来鉴定与GAPD-S和FSC1结合的其他蛋白质，以确定这些蛋白质和我们之前鉴定的其他蛋白质如何组装成纤维鞘。这些蛋白包括男性生殖细胞特异性己糖激酶1 （HK1-S）、谷胱甘肽s转移酶（GSTM5）、AKAP110和两种未知蛋白。基因靶向也被用于生产Fsc1基因敲除小鼠，这些小鼠由于鞭毛结构和功能的破坏而被预测为不育。它们将与表达FSC1的转基因小鼠杂交，这些FSC1带有点突变，可以消除RII&#61474和RII&#61474的结合，或者导致PKA异构体结合的开关。转基因将受到仅在生精细胞中表达的启动子的控制。这些动物模型将允许在体内研究FSC1的功能和不同PKA亚型在精子功能中的作用，以及分析akap和PKA之间的相互作用。我们使用基因靶向来破坏受精素的表达，这是一种精子表面分子，是参与细胞-细胞相互作用的ADAM（一种崩解素和金属蛋白酶结构域）蛋白家族的原型（合作者Myles和Primakoff）。受精卵二聚体参与精卵相互作用，并与卵表面的整合素结合。雄性不育小鼠能产生正常数量的活动精子，并表现出正常的交配行为，但却不能生育，因为进入输卵管的精子很少，而进入输卵管的精子又不能与卵子结合。似乎金属蛋白酶活性的丧失改变了精子与输卵管的相互作用，而崩解素活性的丧失破坏了精子与卵子的结合。我们还使用基因靶向来破坏精蛋白1和2的表达，这是另外两种重要的精子蛋白（合作者Hecht）。精蛋白是一种高度基本的核蛋白，在减数分裂后取代组蛋白，被认为是精细胞（一种单倍体且缺乏核小体的细胞类型）中DNA压缩所必需的。假设一个或两个鱼精蛋白基因的破坏会导致纯合子雄性核压实异常和不育。

Summary of Work: The long-term goals are to identify and characterize key mechanisms that control development or function of germ cells in the male. The approaches being used are to identify genes expressed specifically in male germ cells, to use transgenic mice and in vitro assays to identify promoter elements and transcription factors regulating their expression, and to apply the gene knockout approach to define the roles of the proteins they encode. Many genes are expressed only in male germ cells and we are focusing on a few likely to be important in gamete development or function. We isolated the mouse and human genes for a key isozyme in the glycolytic pathway, glyceraldehyde 3-phosphate dehydrogenase (GAPD-S), that is expressed only in male germ cells and believed to have a key role in regulating generation of ATP required for fertilization. Antibodies prepared to synthetic peptides localized GAPD-S to the fibrous sheath of the sperm flagellum in mouse and human. Yeast two-hybrid screens identified a WW-domain protein (FBP11) which binds to a proline-rich region of mouse GAPD-S. We are mapping the binding domains on each protein and testing the hypothesis that FBP11 anchors GAPD-S to the fibrous sheath. In addition, molecular modeling studies indicate that residues surrounding the substrate-binding pocket may account for differences in the effects of the inhibitor on the somatic and germ cell isozymes (collaborator Bienstock). Human recombinant GAPD-S is being expressed for use in NMR studies to examine the interaction of GAPD-S with its natural substrate, natural cofactor, and reproductive toxicants (collaborator London). One of these is (S)-3-chlorolactaldehyde, a metabolite of the industrial solvent epichlorohydrin that appears to act as a competitive inhibitor of substrate binding to GAPD-S. Furthermore, a Gapds gene-targeting construct is being prepared to test the hypothesis that sperm from knockout mice will be unable to produce the ATP necessary to achieve hyperactivated motility and will be unable to fertilize eggs (collaborators Bunch and O?Brien).We demonstrated previously that fibrous sheath component 1 (FSC1) was the major cytoskeletal protein in the sperm flagellum and that GAPD-S and FSC1 co-localize in this region of the mouse sperm. To study their possible interactions and the role of FSC1, the Fsc1 cDNA and gene were isolated and yeast two-hybrid assays were used to identify associated proteins. FSC1 was found to be a protein kinase A (PKA) anchoring protein (AKAP), suggesting that PKA anchored to the fibrous sheath participates in protein prosphorylation events essential for the activation of sperm motility. Yeast two-hybrid assays, alanine and valine scanning-mutagenesis, and pull-down assays were used to define the amino acids responsible for specificity of binding of different regulatory subunits of the PKA tetramer. RI-specific and dual RI/RII-specific binding motifs identified in FSC1 were the first to be characterization in an AKAP. It was found that hydrophobic amino acids at three consensus positions on AKAPs are required for PKA binding and that specificity of PKA binding is determined by the size of the aliphatic side-chain on the amino acid in the middle position. This was verified by introducing point mutations into these motifs to switch between RI-specific, RII-specific, and RI/RII dual-specific binding. These findings represent an important advance in understanding the relationship between the primary sequence and the three-dimensional spatial distribution of residues within the amphipathic -helix of AKAP anchoring domains that determine PKA binding specificity. They are also significant for advancing the understanding of molecular mechanisms involved in PKA subtype localization within cells.Other studies are using yeast two-hybrid screens to identify additional proteins that bind to GAPD-S and to FSC1 to determine how these and other proteins we identified previously assemble into a fibrous sheath. The proteins include male germ cell-specific isoforms of hexokinase 1 (HK1-S), glutathione S-transferase (GSTM5), AKAP110, and two unknown proteins. Gene targeting is also being used to produce Fsc1 gene knockout mice that are predicted to be infertile due to disruption of flagellar structure and function. They will be crossed with transgenic mice that express FSC1 with point mutations that abolish RI and RII binding or that result in switches in PKA isoform binding. The transgenes will be under the control of a promoter expressed only in spermatogenic cells. These animal models will allow in vivo studies of the function of FSC1 and of the role of different PKA isoforms in sperm function, as well as the analysis of the interactions between AKAPs and PKAs.We used gene targeting to disrupt expression of fertilin , a sperm surface molecule that is the archetype of the ADAM (a disintegrin and metalloprotease domain) family of proteins involved in cell-cell interactions (collaborators Myles and Primakoff). The fertilin : dimer participates in sperm-egg interaction and binds to a 61 integrin on the egg surface. Male fertilin  knockout mice produce typical numbers of motile sperm and show normal mating behavior, but are infertile because few sperm enter the oviduct and those that do are unable to bind to eggs. It appears that loss of metalloprotease activity alters sperm-oviduct interaction and that loss of disintegrin activity disrupts sperm-egg binding. We also are using gene targeting to disrupt expression of protamines 1 and 2, two other important sperm proteins (collaborator Hecht). The protamines are highly basic nuclear proteins that replace the histones following meiosis and are thought to be essential for DNA compaction in spermatids, a cell type that is haploid and lacks nucleosomes. It is hypothesized that disruption of one or both protamine genes will lead to abnormal nuclear compaction and infertility in homozygous males.