Analysis of the primary spermatocytes during spermatogenesis in the mouse.

(1)制备针对原代精母细胞的单克隆抗体。利用间接免疫荧光法和免疫电镜技术，证实了三种抗体可识别小鼠睾丸中不同阶段精母细胞的表面抗原。抗体10B3识别粗线素中晚期（VI-X）的精母细胞，抗体10D6标记细线素至粗线素早期（I-IV）的精母细胞，抗体12F7标记后期（VII-VIII）的精母细胞。(2)抗体10D6和12F7也能识别精子头表面的特异性位点。为了观察抗体对受精的影响，我们将抗体添加到体外受精系统的培养基中，但没有得到明显的效果。(3)用Western blotting对三种抗体识别的抗原进行鉴定。抗体10D6和12F7没有检测到任何斑点，而抗体10B3通过二维Western blotting标记了一个斑点（MW 50kD,pI5.5）。(4)我们尝试培养生精细胞，包括精母细胞和性腺细胞。然而，没有得到显著的结果。(5)精母细胞在成熟过程中从精小管的基底室向腔室移动，细胞骨架蛋白如肌动蛋白丝可能影响精母细胞的运动。因此，我们研究了肌动蛋白丝和肌动蛋白结合蛋白在睾丸中的分布。因此，肌动蛋白丝的分布在出生后发育过程中发生了变化。此外，在精子发生受到干扰的隐睾中，肌动蛋白丝的排列也被破坏。这些结果提示肌动蛋白丝的分布与生精活性之间的关系。

(1) Monoclonal antibodies were raised against primary spermatocytes. Using indirect immunofluorescence assay and immunoelectron microscopy, it has confirmed that three antibodies recognize surface antigens of the different stages of spermatocytes in the mouse testis. Antibody 10B3 recognizes spermatocytes during middle to late pachytene stages (VI-X) , antibody 10D6 stains spermatocyte during leptotene to early pachytene stages (I-IV) and antibody 12F7 labeled spermatocytes during late stages(VII-VIII).(2) Antibodies 10D6 and 12F7 also recognized specific sites of the surface of the sperm head. To see the effects of the antibodies on the fertilization, the antibodies were added to the culture medium of the in vitro fertilization system, but obvious effects were not obtained.(3) Antigens recognized by the three antibodies were characterized by Western blotting. Antibodies 10D6 and 12F7 failed to detect any spot, whereas antibody 10B3 labeled one spot (MW 50kD,pI5.5) by two-dimensional Western blotting.(4) We tried to culture spermatogenic cells including spermatocytes and gonocytes. However, significant results were not obtained.(5) Spermatocytes move from the basal to adluminal compartment in the seminiferous tubule during maturation, and cytoskeletal proteins such as actin filaments might influence the movement of the spermatocytes. Thus we investigated the distribution of actin filaments and actin-binding proteins in the testis. As a result, the distribution of actin filaments changed during postnatal development. Furthermore, in the cryptorchid testis, in which spermatogenesis was disturbad, the arrangement of actin filaments was also disrupted. These results suggest the relation between the distribution of the actin filaments and spermatogenic activity.