Cellular localization and age-dependent changes in mRNA for cyclic adenosine 3',5'-monophosphate-dependent protein kinases in rat testis

Gonadotropin activation of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinases plays an important role in the regulation of testicular function. This study was undertaken to establish the expression of various subunits of cAMP-dependent protein kinases in different testicular cell types as well as during sexual maturation. RNA was extracted from cultured Sertoli cells, cultured peritubular cells, germ cells (pachytene spermatocytes, round spermatids), tumor Leydig cells, as well as whole testis from rats of various ages. Messenger RNA levels were studied by Northern analysis using available cDNA probes. The regulatory subunit (R) designated RII51 was found to be predominantly expressed in cAMP-stimulated Sertoli cells and tumor Leydig cells. Much lower levels were found in cultured peritubular cells and germ cells. A 2.9- and 3.2-kb mRNA for the RI subunit were found at about similar levels in all cell types, whereas the smaller 1.7-kb mRNA was expressed in high levels in germ cells. Also, the catalytic subunit (C) of cAMP-dependent protein kinase, designated C alpha, was expressed in all cell types; the highest mRNA levels for this subunit were found in germ cells and in tumor Leydig cells. The 1.7-kb mRNA for androgen-binding protein (ABP) was abundant in cAMP-stimulated Sertoli cells and was not present in other cell types of the testis. Furthermore, the cellular localization of the cAMP-dependent protein kinase subunits was also supported by developmental studies. The mRNA level of the RII51 3.2-kb species was relatively constant until Day 30, after which there was a tendency to decrease. A 1.6-kb message first appeared at greater ages. The mRNA for the smaller 1.7-kb species of RI, as well as the C alpha, showed a significant increase during development, supporting an enrichment of these mRNAs in germ cells. Messenger RNA levels for ABP were not detected in testis from 5- to 10-day-old rats but increased up to Day 30. After this age, mRNA for ABP revealed an age-dependent decrease, which parallels the relative increase of germ cells in the testis. In summary, these results demonstrate a clear pattern of cellular localization of the various mRNA species for subunits of the cAMP-dependent protein kinase in the rat testis.     

RNF151, a testis-specific RING finger protein, interacts with dysbindin

RING finger proteins play important roles in spermatogenesis. Here, we report that a novel RING finger protein RNF151, with a C3HC4-type RING finger domain, a putative nuclear localization signal (NLS), and a TRAF-type zinc finger domain, was exclusively expressed in the mouse testis and developmentally regulated during spermatogenesis. While RNF151 mRNA was present in round spermatids, its protein was expressed in elongating spermatids of the stage VIII-IX seminiferous tubules. The NLS together with the RING domain were necessary and sufficient for the nuclear localization of RNF151-EGFP in transfected cells. Yeast two-hybrid screening identified the physical interaction of mouse RNF151 and dysbindin, which was confirmed by the co-immunoprecipitation of the proteins and by their co-localization in intact cells. As dysbindin has lately been shown to be involved in membrane biogenesis and fusion, a key process for acrosome formation, we propose that RNF151 may play a role in acrosome formation.     

RNF17, a component of the mammalian germ cell nuage, is essential for spermiogenesis

Nuages are found in the germ cells of diverse organisms. However, nuages in postnatal male germ cells of mice are poorly studied. Previously, we cloned a germ cell-specific gene named Rnf17, which encodes a protein containing both a RING finger and tudor domains. Here, we report that RNF17 is a component of a novel nuage in male germ cells--the RNF17 granule, which is an electron-dense non-membrane bound spherical organelle with a diameter of 0.5 mum. RNF17 granules are prominent in late pachytene and diplotene spermatocytes, and in elongating spermatids. RNF17 granules are distinguishable from other known nuages, such as chromatoid bodies. RNF17 is able to form dimers or polymers both in vitro and in vivo, indicating that it may play a role in the assembly of RNF17 granules. Rnf17-deficient male mice were sterile and exhibited a complete arrest in round spermatids, demonstrating that Rnf17 encodes a novel key regulator of spermiogenesis. Rnf17-null round spermatids advanced to step 4 but failed to produce sperm. These results have shown that RNF17 is a component of a novel germ cell nuage and is required for differentiation of male germ cells.     

SRG4在出生后小鼠睾丸及隐睾中的表达特性

研究小鼠生精新基因SRG4在出生后小鼠睾丸及手术隐睾中的表达特性, 为了解SRG4在精子发生中的作用奠定基础。取出生后1, 3, 12 w小鼠睾丸进行免疫组化检测, 观察SRG4蛋白在出生后小鼠不同发育阶段睾丸中的表达; 制备单侧手术隐睾模型, 取术后0~18 d 的隐睾组织进行半定量RT-PCR检测, 观察SRG4 mRNA在隐睾病变过程中的表达变化, 并对隐睾术后18 d 睾丸进行组织原位杂交分析。免疫组化分析结果表明, SRG4蛋白在出生1 w的小鼠睾丸中几乎检测不到, 在出生3 w的小鼠睾丸中有明显表达, 在出生12 w的小鼠中大量表达, 主要分布在精母细胞和圆形精子细胞胞浆及胞膜, 呈不均匀分布。半定量RT-PCR结果发现, SRG4 mRNA在小鼠隐睾术后0~6 d表达没有明显下调, 9 d 开始表达下调, 第18 d表达最低。组织原位杂交结果表明, 术后18 d隐睾睾丸生殖细胞大量凋亡, 精曲小管中仅见到个别的SRG4阳性信号, 而对照则不受影响。上述结果说明, SRG4蛋白表达受小鼠生长发育调控; 隐睾模型中, 随着生殖细胞的大量凋亡, SRG4基因表达下调, 提示SRG4基因可作为一个精子发生特定阶段的分子标记用以研究精子发生过程。