Meiotic differentiation of germinal cells in three-week cultures of whole cell population from rat seminiferous tubules
Meiotic differentiation of germinal cells in three-week cultures of whole cell population from rat seminiferous tubules
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DOI：
10.1095/biolreprod59.2.379
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发表时间：
1998-08-01
影响因子：
3.6
通讯作者：
Durand, P
中科院分区：
文献类型：
--
作者：
Hue, D;Staub, C;Durand, P
本研究的目的是建立一个培养系统，使大鼠精子发生的大部分减数分裂阶段在体外发生。为此，通过三个标准来监测生精细胞的分化：1)通过逆转录聚合酶链反应（RT-PCR）检测厚质精母细胞（磷酸化蛋白p19 [p19]和睾丸特异性组蛋白TH2B）或圆形精母细胞（过渡蛋白1 [TP1]和过渡蛋白2 [TP2]）中特异性高水平表达的基因的表达；2)倍性分析；生殖细胞的细胞学和免疫细胞化学研究。在第一个实验中，我们测定了产后18 - 60天大鼠整个睾丸中p19:TP1和TH2B:TP2 mrna相关PCR产物的比例变化，并将这些结果与这一时期各种类型生精细胞的顺序出现联系起来。在第二次试验中，我们的目标是在23至25日龄大鼠的精管培养系统中再现在体内观察到的变化。大鼠睾丸提取物的p19:TP1和TH2B:TP2比值在产后32 ~ 40天，即睾丸内圆形精子数量越来越多的时期显著下降。当输精管在双室室中播种时，在3周的培养期间，细胞存活率保持在接近总细胞的70%。在培养的第一周，p19:TP1和TH2B:TP2比值均下降。这是由于p19和TH2B mrna水平的降低，以及TP1和TP2相对量的增加。这些变化与培养中1C细胞群的出现有关。培养物的组织学检查表明，在本研究条件下，IV-VI期5-溴-2'-脱氧尿苷标记的厚线精细胞能够分化为次级精细胞，然后分化为圆形精细胞。
The aim of the present study was to set up a culture system allowing most of the meiotic phase of rat spermatogenesis to occur in vitro. For that purpose, the differentiation of spermatogenic cells was monitored by three criteria: 1) examination of expression of genes specifically expressed at a high level in pachytene spermatocytes (the phosphoprotein p19 [p19] and the testis-specific histone TH2B) or in round spermatids (transition protein 1 [TP1] and transition protein 2 [TP2]) by reverse transcription-polymerase chain reaction (RT-PCR); 2) ploidy analysis; and 3) cytological and immunocytochemical study of the germ cells. In the first trial, we determined the changes in the ratios of p19:TP1 and TH2B:TP2 mRNA-related PCR products in the whole testis of rats between 18 and 60 days postpartum and related those results to the sequential appearance of the various types of spermatogenic cells during that period. In the second trial, our aim was to reproduce, in a culture system using seminiferous tubules from 23- to 25-day-old rats, the changes observed in vivo. The p19:TP1 and TH2B:TP2 ratios decreased dramatically in testicular extracts of rats between 32 and 40 days postpartum, i.e., at the time period during which round spermatids become more and more numerous in the testis. When seminiferous tubules were seeded in bicameral chambers, cell viability remained close to 70% of total cells throughout the 3-wk culture period. Both p19:TP1 and TH2B:TP2 ratios decreased during the first week of culture. This was attributable to a decrease in the levels of p19 and TH2B mRNAs and also to an enhancement in the relative amounts of TP1 and TP2. These changes were correlated with the appearance of a 1C cell population in the culture. Histological examination of the culture demonstrated that under the conditions of the present study, 5-bromo-2'-deoxyuridine-labeled pachytene spermatocytes of stages IV-VI were able to differentiate into secondary spermatocytes, then into round spermatids.