Caldwell K A, Wiltshire T, Handel M A
Department of Zoology, University of Tennessee, Knoxville, USA.
Mol Reprod Dev. 1996 Apr;43(4):403-13. doi: 10.1002/(SICI)1098-2795(199604)43:4<403::AID-MRD1>3.0.CO;2-T.
The goals of this work were to create germ-cell-stage-specific cDNA libraries from mouse spermatogenic cells and to employ a novel two-step genetic screen to identify gene sequences present during the critical meiotic stage of spermatogenesis. Highly enriched germ-cell fractions were prepared from adult and juvenile mouse testes, and purity of these fractions was extensively analyzed by light and electron microscopy. Standard techniques were used to prepare cDNA libraries from populations of mixed leptotene and zygotene (L/Z) spermatocytes, pachytene (P) spermatocytes, and round spermatids. These libraries were analyzed with respect to representation of sequences from ubiquitously expressed genes, and from genes expressed at specific germ-cell stages as well as from genes expressed in testicular somatic cells. For the first step of the screening procedure, testicular cDNA was prepared from mutant mice carrying the T(X;11)38H chromosomal translocation that causes spermatogenic arrest at early meiotic prophase. This mixed cDNA probe was used to screen the libraries from L/Z and P spermatocytes to detect sequences failed to hybridize. The clones identified were characterized for ability to hybridize to various germ-cell-specific cDNAs to verify that they represented sequences present in normal spermatogenic meiotic cells. These clones were then subjected to a second screening with another mutant probe; this time the cDNA probe was from testes of sterile mice bearing the T(X;16)16H chromosomal translocation that causes spermatogenic arrest at late meiotic prophase. This screen identified 27 clones that were not represented in testicular cDNA from T38-bearing mice or from T16-bearing mice. These clones may represent sequences essential for normal completion of the genetic events of meiosis during spermatogenesis. Likewise, the secondary screen identified 19 clones that were not represented in testicular cDNA from T38-bearing mice but were represented in testicular cDNA of T16-bearing mice. These clones are thus gene sequences present in spermatogenic cells during the time from early meiotic prophase to mid-to-late prophase. This strategy represents the first use of genetic aberrations in differential screening to identify genes expressed at specific times during mammalian spermatogenesis.
这项工作的目标是从小鼠生精细胞创建生殖细胞阶段特异性的cDNA文库，并采用一种新型的两步遗传筛选方法来鉴定精子发生关键减数分裂阶段存在的基因序列。从成年和幼年小鼠睾丸中制备高度富集的生殖细胞组分，并通过光学和电子显微镜对这些组分的纯度进行了广泛分析。使用标准技术从混合的细线期和偶线期（L/Z）精母细胞、粗线期（P）精母细胞和圆形精子细胞群体中制备cDNA文库。对这些文库进行了分析，以了解来自普遍表达基因的序列、特定生殖细胞阶段表达的基因以及睾丸体细胞中表达的基因的代表性。在筛选程序的第一步中，从携带T(X;11)38H染色体易位的突变小鼠制备睾丸cDNA，该易位导致减数分裂前期早期精子发生停滞。这个混合的cDNA探针用于筛选L/Z和P精母细胞的文库，以检测未能杂交的序列。对鉴定出的克隆进行杂交各种生殖细胞特异性cDNA的能力表征，以验证它们代表正常精子发生减数分裂细胞中存在的序列。然后用另一种突变探针对这些克隆进行第二次筛选；这次的cDNA探针来自携带T(X;16)16H染色体易位的不育小鼠的睾丸，该易位导致减数分裂前期后期精子发生停滞。这次筛选鉴定出27个在携带T38小鼠或携带T16小鼠的睾丸cDNA中未出现的克隆。这些克隆可能代表精子发生过程中减数分裂遗传事件正常完成所必需的序列。同样，二次筛选鉴定出19个在携带T38小鼠的睾丸cDNA中未出现但在携带T16小鼠的睾丸cDNA中出现的克隆。因此，这些克隆是从减数分裂前期早期到中期至后期存在于生精细胞中的基因序列。这种策略代表了首次在差异筛选中利用遗传畸变来鉴定哺乳动物精子发生过程中特定时间表达的基因。
