Gene Expression In Spermatogenic Cells

长期目标是确定和描述控制男性生殖细胞发育和功能的内在遗传程序的关键组成部分。目前应用的方法是鉴定在男性生殖细胞中特异性表达的基因，使用基因敲除方法来确定它们编码的蛋白质的作用，使用酵母双杂交试验和删除诱变来鉴定雄性配子发育所需的蛋白质-蛋白质相互作用，以及制备和使用抗血清来确定特定基因产物的时空分布。许多基因只在男性生殖细胞中表达，我们关注的是一些编码蛋白，它们的功能可以测定，可能对配子的发育或功能很重要。(1)一个项目涉及糖酵解途径中的关键酶。甘油醛3-磷酸脱氢酶（GAPD）基因在男性生殖细胞中表达是沉默的，而是表达GAPDS，一种生精细胞特异性同源物。我们克隆了小鼠和人GAPDS基因，制备了小鼠GAPDS特异性肽序列抗体，并将GAPDS定位在精子鞭毛纤维鞘上。我们假设这种酶在产生精子运动所需的ATP中起着关键作用。我们产生了GAPDS基因敲除小鼠，我们发现精子活力严重受损，导致雄性不育。重组GAPDS是为了研究该酶与其天然底物和辅因子的相互作用。生殖毒理学研究表明，GAPD和GAPDS具有不同的底物结合特性。某些化合物在低于GAPD的浓度下作为底物与GAPDS结合的竞争性抑制剂。我们的分子模型研究表明，GAPDS和GAPD在底物结合袋附近的残留差异可能解释了毒物对精子的影响。这也表明GAPDS是开发高度特异性男性避孕药的潜在目标。(2)另一个项目涉及纤维鞘的组装和功能，这是精子鞭毛的主要细胞骨架结构。酵母双杂交筛选发现了一个ww结构域蛋白（FBP3），该蛋白与小鼠GAPDS富含脯氨酸的区域结合。我们正在绘制每个蛋白上的结合域，并测试FBP3将GAPDS锚定在纤维鞘上的假设。我们证明了GAPDS与纤维鞘结合，这是精子鞭毛的细胞骨架结构。纤维鞘的主要结构蛋白是由AKAP4基因编码的蛋白激酶a （PKA）锚定蛋白（AKAP）。我们使用酵母双杂交实验来鉴定与AKAP4相关的蛋白，并证实PKA与AKAP4结合，并可能调节camp依赖性蛋白磷酸化，这是激活精子运动所必需的。利用酵母双杂交、丙氨酸和缬氨酸扫描诱变和拉下实验确定了AKAP4中与PKA四聚体调控亚基结合的氨基酸。鉴定了ri - α特异性和双ri - α / ri - α特异性结合基序。我们发现PKA结合需要三个一致位置的疏水氨基酸，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，在ri - α特异性、ri - α特异性和ri - α / rii - α特异性双结合之间切换，证实了这一点。这些发现促进了我们对决定PKA结合特异性的AKAP锚定结构域的一级序列和两亲α -螺旋内残基的三维空间分布之间关系的理解。它们对于理解PKA亚型在细胞内定位的分子机制也具有重要意义。此外，酵母双杂交筛选用于鉴定纤维鞘中与AKAP4结合的其他蛋白质。我们发现AKAP3和两个仅在生精细胞中表达的新蛋白也与AKAP4结合。利用Cre/ loxp介导的基因突变产生Akap4基因敲除小鼠，发现雄性由于鞭毛结构和功能的破坏而不育。(3)另一个项目使用基因靶向来破坏编码蛋白1和2的基因的表达，蛋白1和2是减数分裂后取代组蛋白的高度碱性核蛋白。它们被认为对精子细胞中的DNA压缩至关重要，精子细胞的细胞核是单倍体，缺乏核小体。精子通过细胞质桥连接，通过细胞质桥它们共享mRNA和蛋白质。我们发现，鱼精蛋白1或2基因拷贝的破坏会导致精子结构和功能的改变，并且无法通过雄性嵌合体的种系传递突变等位基因。我们发现，在所有精子中，蛋白质共享导致蛋白质数量减少一半，导致精子发生过程中核压实缺陷。这似乎是第一次观察到单倍体缺陷导致哺乳动物遗传破坏。(4)另一个项目是基于其他人的发现，p55CDC是酵母Cdc20的哺乳动物同源物，Cdc20是一种与环小体/后期促进复合物（APC）相关的蛋白质，对APC依赖性蛋白水解至关重要。在培养的细胞中，p55CDC位于有丝分裂开始时的着丝点，在中期/后期的纺锤体极点，以及从后期到末期的纺锤体赤道。我们发现，改变p55CDC保守的第7个WD40基序的点突变消除了这些动力学，并导致蛋白质在核周内质网中积累。这表明一种结合WD40基序的蛋白参与了p55CDC的定位。以第7个WD40基序为诱饵，在酵母双杂交筛选中鉴定出WDAP1 （WD40适配器蛋白1）为p55cdc结合因子。目前的研究正在确定ADAP1是否在男性减数分裂过程中调节p55CDC的功能。

The long-term goals are to identify and characterize key components of the intrinsic genetic program that controls development and function of male germ cells. The approaches being applied are to identify genes expressed specifically in male germ cells, use the gene knockout approach to define the roles of the proteins they encode, use yeast two-hybrid assays and deletion mutagenesis to identify protein-protein interactions essention for development of the male gamete, and preparation and use of antisera to determine the temporal-spatial distribution of specific gene products. Many genes are expressed only in male germ cells and we are focusing on a few encoding proteins whose functions can be assayed and are likely to be important in gamete development or function. (1) One project involves a key enzyme in the glycolytic pathway. The gene for glyceraldehyde 3-phosphate dehydrogenase (GAPD) expressed throughout the body is silent in male germ cells and instead GAPDS, a spermatogenic cell-specific homolog, is expressed. We cloned the genes for mouse and human GAPDS, prepared antibodies to peptide sequences specific to mouse GAPDS, and localized GAPDS to the fibrous sheath of the sperm flagellum. We hypothesized that the enzyme has a key role in generating the ATP required for sperm motility. GAPDS knockout mice were generated and we found that sperm motility was severely compromised, causing the males to be infertile. Recombinant GAPDS was produced to study the interaction of the enzyme with its natural substrate and cofactor. Reproductive toxicology studies by others suggested that GAPD and GAPDS have different substrate -binding characteristics. Certain compounds act as competitive inhibitors of substrate binding to GAPDS at a lower concentration than GAPD. Our molecular modeling studies indicate that residue differences between GAPDS and GAPD in the vicinity of the substrate-binding pocket probably account for effects of the toxicants on sperm. This also suggests that GAPDS is a potential target for development of a highly specific male contraceptive. (2)Another project involves the assembly and function of the fibrous sheath, a major cytoskeletal structure in the sperm flagellum. Yeast two-hybrid screens identified a WW-domain protein (FBP3) which binds to a proline-rich region of mouse GAPDS. We are mapping the binding domains on each protein and testing the hypothesis that FBP3 anchors GAPDS to the fibrous sheath. We demonstrated that GAPDS binds to the fibrous sheath, a cytoskeletal structure in the sperm flagellum. The major structural protein of the fibrous sheath is a protein kinase A (PKA) anchoring protein (AKAP) encoded by the AKAP4 gene. We used yeast two-hybrid assays to identify proteins that associate with AKAP4 and confirmed that PKA binds to AKAP4 and may regulate cAMP-dependent protein-phosphorylation essential for activation of sperm motility. Yeast two-hybrid assays, alanine and valine scanning-mutagenesis, and pull-down assays were used to define the amino acids of AKAP4 responsible for the binding of regulatory (R) subunits of the PKA tetramer. RI-alpha-specific and dual RI-alpha/RII-alpha-specific binding motifs were identified. It was found that hydrophobic amino acids at three consensus positions 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-alpha-specific, RII-alpha-specific, and dual RI-alpha/RII-alpha-specific binding. These findings advance our understanding of the relationship between the primary sequence and the three-dimensional spatial distribution of residues within the amphipathic alpha-helix of AKAP anchoring domains that determine PKA binding specificity. They are also significant for understanding the molecular mechanisms involved in PKA subtype localization within cells. In addition, yeast two-hybrid screens were used to identify other proteins that bind to AKAP4 in the fibrous sheath. We found that AKAP3 and two novel proteins expressed only in spermatogenic cells also bind to AKAP4. Cre/loxP-mediated gene mutation was used to produce Akap4 gene knockout mice and the males were found to be infertile due to disruption of flagellar structure and function. (3) Another project used gene targeting to disrupt expression of the genes encoding protamines 1 and 2, highly basic nuclear proteins that replace histones following meiosis. They are thought essential for DNA compaction in spermatids, whose nuclei are haploid and lack nucleosomes. Spermatids are connected by cytoplasmic bridges, through which they share mRNA and protein. We found that disruption of one copy of a gene for either protamine 1 or 2 led to altered sperm structure and function and failure to transmit the mutant allele through the germ line of male chimeras. We found that protein sharing leads to reduction in amount of protein by one-half in all spermatids, resulting in defective nuclear compaction during spermiogenesis. This appears to be the first observation of haplo-insufficiency leading to disruption of genetic inheritance in mammals. (4) An addition project is based on findings by others that p55CDC is the mammalian homologue of yeast Cdc20, a protein which associates with the cyclosome/anaphase-promoting complex (APC) and is essential for APC-dependent proteolysis. In cultured cells, p55CDC is located at the kinetochores at the beginning of mitosis, at the spindle poles through the metaphase/anaphase transition, and at the spindle equator from anaphase to telophase. We found that point mutations altering the conserved seventh WD40 motif of p55CDC abolished these dynamics and causes the protein to accumulate in the perinuclear endoplasmic reticulum. This suggested that a protein that binds to the WD40 motif is involved in localizing p55CDC. By using the seventh WD40 motif as bait in a yeast two-hybrid screen, WDAP1 (WD40 adapter protein 1) was identified as a p55CDC-binding factor. Current studies are determing if ADAP1 regulates the function of p55CDC during meiosis in the male.