Expression of a Y-box protein, YB2/RYB-a, precedes protamine 2 expression during spermatogenesis in rodents.

Y-box binding proteins, a large family of proteins, are involved in a variety of functions. The present study describes the expression of YB2, a rat Y-box binding protein, and/or RYB-a, an alternatively spliced product of the YB2 gene during spermatogenesis. YB2/RYB-a is thought to be the rat orthologue of mouse Y-box protein 3 (MSY3). An antibody which recognizes YB2/RYB-a was developed and applied in an immunochemical study of rat and mouse testes. We also carried out an in-situ hybridization study and Northern blot analysis of YB2/RYB-a and protamine 2 mRNA expression. Both YB2/RYB-a mRNA and the proteins appeared in prepubertal mouse testes, prior to the expression of the mouse protamine 2 mRNA. The mRNA and protein were present at high levels in spermatocytes, decreased in round to elongated spermatids, and were absent in spermatozoa. Since the protamine 2 mRNA was present at high levels in round and elongating spermatids, the proposed function of the YB2/RYB-a protein as a translational repressor of the mRNA was supported in mouse. The level and localization of YB2/RYB-a mRNA and protein expression in the rat testis was comparable to that in mouse testis, although rat testis is known to express a very low level of protamine 2, but is also likely to affect the expression of other proteins (including protamine 1) during spermatogenesis.     

Compensatory upregulation of myelin protein zero-like 2 expression in spermatogenic cells in cell adhesion molecule-1-deficient mice

The cell adhesion molecule-1 (Cadm1) is a member of the immunoglobulin superfamily. In the mouse testis, Cadm1 is expressed in the earlier spermatogenic cells up to early pachytene spermatocytes and also in elongated spermatids, but not in Sertoli cells. Cadm1-deficient mice have male infertility due to defective spermatogenesis, in which detachment of spermatids is prominent while spermatocytes appear intact. To elucidate the molecular mechanisms of the impaired spermatogenesis caused by Cadm1 deficiency, we performed DNA microarray analysis of global gene expression in the testis compared between Cadm1-deficient and wild-type mice. Out of the 25 genes upregulated in Cadm1-deficient mice, we took a special interest in myelin protein zero-like 2 (Mpzl2), another cell adhesion molecule of the immunoglobulin superfamily. The levels of Mpzl2 mRNA increased by 20-fold and those of Mpzl2 protein increased by 2-fold in the testis of Cadm1-deficient mice, as analyzed with quantitative PCR and western blotting, respectively. In situ hybridization and immunohistochemistry demonstrated that Mpzl2 mRNA and protein are localized in the earlier spermatogenic cells but not in elongated spermatids or Sertoli cells, in both wild-type and Cadm1-deficient mice. These results suggested that Mpzl2 can compensate for the deficiency of Cadm1 in the earlier spermatogenic cells.     

小鼠精子发生相关蛋白3基因在小鼠生精细胞的特异表达及对HEK 293T细胞凋亡和自噬的影响

目的检测小鼠精子发生相关蛋白3(spata3)基因在小鼠生精细胞的表达情况,并借助过表达细胞模型进一步分析该基因对人胚肾HEK 293T细胞凋亡及自噬的影响,旨在探讨spata3在精子发生过程中的意义。方法分别采用RT-PCR和免疫印迹法检测spata3基因mRNA及蛋白产物在小鼠各组织中的表达;应用免疫组织化学和免疫荧光染色观察SPATA3蛋白在生精细胞中的定位;借助脂质体将真核表达载体Plv-EGFP-2(a)purospata3瞬时转染HEK 293T细胞,进一步在蛋白水平分析细胞凋亡相关蛋白Caspase-3、多聚ADP-核糖聚合酶(PARP)、BAX和Bcl-2及自噬相关蛋白LC3A/B的变化。结果 Spata3基因及其编码产物在小鼠睾丸组织特异表达;粗线期精母细胞和圆形精子细胞的胞质与胞核均有显著SPATA3蛋白的阳性着色,长形精子细胞的胞质也有大量分布;过表达spata3的HEK 293T细胞内活化型Caspase-3和PARP降解产物的含量较对照组差异无显著性,BAX表达量0.815±0.020较裸细胞组0.469±0.012和空载体转染组0.588±0.018均有所增高,Bcl-2含量0.214±0.020低于裸细胞0.507±0.021和空载体转染组0.545±0.024,LC3A/B-Ⅱ的表达量0.741±0.037则显著高于裸细胞组0.136±0.011和空载体转染组0.169±0.012。结论 Spata3基因在小鼠生精细胞特异表达,过表达spata3对HEK 293T细胞凋亡无明显影响,但可以促进细胞自噬。     

低剂量电离辐射诱导小鼠睾丸生精细胞凋亡及P53基因和蛋白表达

目的:研究低剂量电离辐射对小鼠睾丸生精细胞凋亡及 P53 基因和蛋白表达的影响。方法:应用密度梯度离心法分离不同种类生精细胞, 流式细胞术检测其细胞凋亡, 免疫组化法观察生精细胞P53蛋白表达, 原位杂交法观察其 P53 mRNA水平。结果:0.025-0.2 Gy X射线全身照射后, 生精细胞凋亡具有明显的细胞种类规律性。在较低剂量照射(0.025和0.05 Gy)时, 以精原细胞凋亡为主, 随照射剂量增加(0.075-0.2 Gy)逐渐累及精母细胞, 并且前者凋亡率明显高于后者, 很少累及精子细胞和精子。P53蛋白表达主要见于精原细胞和精母细胞, 并且前者阳性率高于后者, 随照射剂量增加, 其阳性率逐渐升高, 而精子细胞和精子阳性率较低; P53 mRNA表达在较低剂量照射(0.025 Gy)时, 主要以精母细胞和精子细胞为主, 随剂量增加(0.05-0.2 Gy)逐渐累及精原细胞。精原细胞和精母细胞 P53 mRNA表达呈明显的剂量依赖性关系, 但精子细胞表现不明显。结论:低剂量电离辐射可选择性诱导小鼠睾丸生精细胞凋亡, 具有明显的剂量和时程效应关系。提示, 这种选择性诱导凋亡调控机制可能与 P53 基因和蛋白表达相关联。     

Differential expression of complement regulatory proteins decay-accelerating factor (CD55), membrane cofactor protein (CD46) and CD59 during human spermatogenesis.

We have examined the distribution of the complement (C) regulatory proteins CD59, membrane cofactor protein (MCP) and decay-accelerating factor (DAF) on mature sperm and compared expression of these proteins in parallel both during spermatogenesis and in the prostate. Enhanced immunoperoxidase staining and radioimmunoassay confirmed that C regulators are differentially expressed on sperm; CD59 was strongly expressed on the surface of acrosome intact sperm while MCP and DAF appear to be located primarily on the inner acrosomal membrane. While the MW of CD59 on sperm is typical of other systems, we confirm that in addition to a novel 40,000-46,000 MW MCP protein, sperm also express a novel 55,000 MW DAF product. Examination of normal testis by immunostaining revealed that although C regulators are differentially expressed within the germinal epithelium, all three proteins were present on the acrosomal region of condensing spermatids. We show that novel, low MW forms of MCP and DAF are expressed in normal testis membranes but are absent from testis membranes obtained from patients undergoing gender reassignment surgery in whom the germinal epithelium is diminished. Novel MW C3 convertase regulators are therefore associated with differentiating germinal epithelium. Typical CD59 components were also present on normal testis membranes confirming that CD59 is acquired during spermatogenesis. We demonstrate that the prostatic epithelium, in addition to MCP, expresses CD59 but not DAF. By comparison with CD59, therefore, our studies suggest that DAF may be acquired only in the testis. Overall, our data suggest that, on leaving the testis, sperm express the repertoire of C regulators required for protection from C during their transit through the male and female reproductive tracts.     

Decay accelerating factor in guinea-pig reproductive organs

Decay accelerating factor (DAF, CD55) expressed in human reproductive organs and gametes is thought to play a pivotal role in protection against autologous complement activation in the genital tract. To further investigate the role of DAF in reproduction, we analysed DAF distribution in reproductive organs using guinea-pigs that express multiple DAF isoforms. In males, significant staining was observed in testis on the elongated spermatids and spermatozoa. Levels of DAF mRNA with a shorter 3' untranslated region were significantly enhanced in testis from 9 weeks of age, indicating the presence of DAF mRNA and protein synthesis of spermatozoa DAF in late haploid germ cells. Epididymal spermatozoa appeared to express DAF on the inner acrosomal membrane as well as over their entire surface. Significant DAF expression was also observed on the epithelium of seminal vesicles from 4 weeks of age, with no increase thereafter in the mRNA. C3 mRNA was not detected in this tissue. In females, DAF was detected on the plasma membranes of oocytes through follicle development and on the apical region of uterine epithelium, although the levels of DAF mRNA in these tissues were low. In addition, DAF was selectively expressed on the apical region of ciliated oviductal epithelial cells. The apical region of the ciliated cells comprising the efferent ductule epithelium was also stained significantly, even at 12 days of age, while other epididymal epithelial cells were hardly stained at any age, suggesting that DAF is constitutively expressed on cilia.     

Histological changes of acrylamide-induced testicular lesions in mice

The effects of a single oral dose of 150 or 100 mg/kg acrylamide on the testis were studied histopathologically in prepubertal and adult mice over a period of 10 days following treatment. In the prepubertal mice, severe testicular damage such as vacuolation and swelling of the round spermatids, necrosis of the late elongated spermatids in stages I to VIII, abnormal meiosis in stage XIX, and a marked cellular exfoliation into the lumen were detected 1 day after administration of the 150 mg/kg dose. On Day 2, the damage was more pronounced. However, the testicular damage was repaired 7 to 10 days after treatment. In the adults, many tubules from stages I to VIII were affected, showing nuclear vacuolation in most of the round spermatids 1 day after administration of the 150 mg/kg dose. Meiosis in stage XIV was normal. In the prepubertal mice, nuclei of the round spermatids in stage I-III, especially step 1 spermatids had degenerated 1 day after administration of the 100 mg/kg dose. In the adults, the round spermatids had degenerated in stages I-II and IV-VIII 1 day after administration of the 100 mg/kg dose. These results suggested that the most vulnerable cell type among the spermatogenic cells was the round spermatid, especially that in the Golgi phase (stage I-III). Other stages and spermatogenic cells were relatively resistant to acrylamide testicular toxicity.     

Cloning of cDNAs and the differential expression of A-type cyclins and Dmc1 during spermatogenesis in the Japanese eel, a teleost fish.

We cloned A-type cyclins (cyclins A1 and A2) and Dmc1 cDNAs from the eel testis. Cyclin A1 mRNA was predominantly expressed in the livers, ovaries, and testes of the eels. In contrast to cyclin A1 mRNA, a very high expression of cyclin A2 mRNA was observed in the brains, livers, kidneys, spleens, ovaries, and testes of the eels. Dmc1 mRNA was predominantly expressed in the testes and ovaries; expression in the brain was also detected. In the eel testis, a few type-A spermatogonia incorporating 5-bromo-2'-deoxyuridine (BrdU) were seen before the initiation of spermatogenesis by hormonal induction. On day 1 after hormonal induction, the number of BrdU-labeled spermatogonia increased remarkably, and after 3 and 6 days, many labeled type-B spermatogonia were also observed. The expression of cyclin A2 increased 1 day after the induction of spermatogenesis and reached a plateau after 6 days, when many type-B spermatogonia with high proliferative activity were found. In contrast, the expression of cyclin A1 mRNA was detected after 9 days, coincident with the first appearance of spermatocytes. Cyclin A1 mRNA was localized in germ cells of all stages, from primary spermatocytes to round spermatids, whereas cyclin A2 mRNA was specifically localized in spermatogonia, secondary spermatocytes, round spermatids, and testicular somatic cells, including Sertoli cells. Dmc1 was localized only in the earlier stages of primary spermatocytes; before this stage, cyclin A1 mRNA was not detectable. Overall, cyclin A2, Dmc1, and cyclin A1 are expressed in spermatogenic cells sequentially before and during meiosis in the eel testis.     

Dynamic changes in intranuclear and subcellular localizations of mouse Prrp/DAZAP1 during spermatogenesis: the necessity of the C-terminal proline-rich region for nuclear import and localization

Mouse Prrp (mPrrp)/DAZAP1 is a mouse ortholog of Xenopus Prrp, which is involved in vegetal pole localization of Vg1 mRNA in oocytes and is highly expressed in the testis. The mouse protein has been reported to be a shuttling protein which localizes in the nucleus of pre-meiotic spermatogenic cells and round spermatids, and shifts its location into the cytoplasm in elongating spermatids, suggesting that mPrrp may be involved in mRNA transport as well as that of the Xenopus ortholog. We reexamined immunohistochemical analyses of mPrrp/DAZAP1 during spermatogenesis utilizing a newly established monoclonal antibody and reconfirmed it to be a shuttling protein. We also carried out new observations that included remarkable intranuclear movement during spermatogenesis. In addition, we found that a long amino acid stretch which spanned over the C-terminal half of the protein was required for the nuclear import. These observations demonstrated dynamic changes in subnuclear and subcellular localization which might reflect specific functions during spermatogenesis.     

层黏连蛋白受体在小鼠睾丸和附睾中的表达

目的 探讨层黏连蛋白受体(LAMR1)在小鼠睾丸和附睾中的表达.方法收集3只正常成年昆明小鼠睾丸和附睾.采用原位杂交和免疫组织化学方法,检测LAMR1 mRNA及蛋白在成年小鼠睾丸和附睾中的分布.结果 LAMR1 mRNA在附睾头和附睾尾表达最强;在睾丸生精细胞中也有表达.免疫组织化学结果显示,LAMR1蛋白从附睾头到...     

Increased protein arginine methylation in chronic hypoxia: role of protein arginine methyltransferases

Asymmetric dimethylarginine (ADMA) is an endogenous inhibitor of nitric oxide synthesis. ADMA is generated by catabolism of proteins containing methylated arginine residues, and its levels are correlated with endothelial dysfunction in systemic cardiovascular diseases. Arginine methylation of cellular proteins is catalyzed by protein arginine methyltransferases (PRMT). The expression and localization of PRMT in the lung has not been addressed. Here, we sought to analyze the expression of PRMT isoforms in the lung and to determine whether PRMT expression is altered during exposure to chronic hypoxia (10% oxygen). Adult mice were exposed to hypoxia for up to 3 wk, and lung tissues were harvested and processed for RT-PCR, Western blotting, immunohistochemistry, and determination of tissue ADMA levels. All PRMT isoforms investigated were detected at the mRNA and protein level in mouse lung, and were localized primarily to the bronchial and alveolar epithelium. In lungs of mice subjected to chronic hypoxia, PRMT2 mRNA and protein levels were up-regulated, whereas the expression of all other PRMT isoforms remained unchanged. This was mainly due to increased expression of PRMT2 in alveolar type II cells, which did not express detectable levels of PRMT2 under normoxic conditions. Consistent with these observations, lung ADMA levels and ADMA/l-Arginine ratios were increased under hypoxic conditions. These results demonstrate that PRMTs are expressed and functional in the lung, and that hypoxia is a potent regulator of PRMT2 expression and lung ADMA concentrations. These data suggest that structural and functional changes caused by hypoxia may be linked to ADMA metabolism.     

Differential expression of multiple isoforms of the ELKS mRNAs involved in a papillary thyroid carcinoma

A novel gene, ELKS, whose 5' portion was fused to the RET gene, was found in a papillary thyroid carcinoma. A cDNA of this gene obtained from a human-brain cDNA library revealed that it encoded a peptide of 948 amino acids, termed ELKSalpha. We identified four other isoforms, which encoded ELKSbeta, ELKSgamma, ELKSdelta, and ELKSepsilon proteins consisting, respectively, of 992, 720, 1088, and 1116 amino acid residues. Analysis of the gene structure revealed that the isoforms were generated by alternative splicing. Isoforms beta, gamma, delta, and epsilon all contain an optional exon (exon14a), but ELKSgamma, -delta, and -epsilon lack exon 1b. ELKSgamma lacks exons 3 to 6. ELKSdelta and -epsilon lack exons 12 and 17; ELKSepsilon contains an optional exon (exon 6a). Analysis by RT-PCR suggested that ELKSalpha and ELKSbeta mRNAs are abundant in the brain, ELKSdelta and ELKSepsilon mRNAs predominate in testis and thyroid, and ELKSepsilon mRNA predominates in other tissues. To prove whether the fusion of different ELKS isoforms to RET (between ELKS coiled-coil domains and the RET kinase domain) could produce chimeric proteins that could be autophosphorylated, we synthesized ELKSgamma-RET, ELKSdelta-RET, and ELKSepsilon-RET fusion proteins in vitro. Immunoblotting with anti-ELKS, anti-RET, and anti-phosphotyrosine antibodies demonstrated that the chimeric proteins were constitutively phosphorylated at tyrosine residues, whereas native RET protein was not. These results indicate that the ELKS gene is alternatively spliced, and that every type of ELKS-RET chimeric protein having oligomerization domains can activate RET's cytoplasmic tyrosine kinase.