Structure and expression of the zebrafish mest gene, an ortholog of mammalian imprinted gene PEG1/MEST

PEG1/MEST is a paternally expressed gene in placental mammals. Here, we report identification of zebrafish (Danio rerio) gene mest, an ortholog of mammalian PEG1/MEST. Zebrafish mest encodes a polypeptide of 344 amino acids and shows a significant similarity to mammalian orthologs. Zebrafish mest is present as a single copy in the zebrafish genome and is closely linked to copg2 as in mammals. It is notable that 10 of 11 intron positions in mest are conserved among mammalian PEG1/MEST genes, indicating that the genomic organization and linkage between mest and copg2 loci was established in ancient vertebrates. Zebrafish mest is expressed in blastula, segmentation, and larval stages, exhibiting gradually increased expression as the development proceeds. Allelic expression analysis in hybrid larvae shows that both parental alleles are transcribed. We also observed one-codon alternative splicing involving an alternative usage of the two consecutive splice acceptors of intron 1, generating two protein isoforms with different lengths of a single amino acid.     

T-SP1: a novel serine protease-like protein predominantly expressed in testis

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends (RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue.     

Characterization of human RhCG and mouse Rhcg as novel nonerythroid Rh glycoprotein homologues predominantly expressed in kidney and testis

In mammals, the Rh family includes the variable Rh polypeptides and invariant RhAG glycoprotein. These polytopic proteins are confined to the erythroid lineage and are assembled into a multisubunit complex essential for Rh antigen expression and plasma membrane integrity. Here, we report the characterization of RhCG and Rhcg, a pair of novel Rh homologues present in human and mouse nonerythroid tissues. Despite sharing a notable similarity to the erythroid forms, including the 12-transmembrane topological fold, the RHCG/Rhcg pair is distinct in chromosome location, genomic organization, promoter structure, and tissue-specific expression. RHCG and Rhcg map at 15q25 of human chromosome 15 and the long arm of mouse chromosome 7, respectively, each having 11 exons and a CpG-rich promoter. Northern blots detected kidney and testis as the major organs of RHCG or Rhcg expression. In situ hybridization revealed strong expression of Rhcg in the kidney collecting tubules and testis seminiferous tubules. Confocal imaging of transiently expressed green fluorescence protein fusion proteins localized RhCG exclusively to the plasma membrane, a distribution confirmed by cellular fractionation and Western blot analysis. In vitro translation and ex vivo expression showed that RhCG carries a complex N-glycan, probably at the (48)NLS(50) sequon of exoloop 1. These results pinpoint RhCG and Rhcg as novel polytopic membrane glycoproteins that may function as epithelial transporters maintaining normal homeostatic conditions in kidney and testis.     

镉对小鼠精母细胞凋亡及附睾内精子质量的影响

采用人工水质染毒的方法,利用透射电镜技术及流式细胞术（FCM）,探讨重金属镉对小鼠精巢内生殖细胞凋亡及附睾内成熟精子质量的影响.结果表明：各试验组小鼠生殖细胞处于凋亡时期的数量显著高于对照组,凋亡时期的生殖细胞超微结构呈现出线粒体空泡、核膜内陷、染色质周缘化及核固缩等形态特征,表明镉容易引起小鼠生殖细胞凋亡;各试验组精子早期凋亡的比例显著高于对照组,而活性精子的比例显著低于对照组（P<0.05）,其中高剂量组（0.10 mmol·L^-1）精子成活率（75.1%）显著低于对照组和其他试验组,而早期凋亡率（22.6%）则显著高于对照组;高剂量组睾丸生殖细胞DNA断裂率（18.2%）及附睾精子断裂率（26.5%）均显著高于对照组（3.3%、5.6 %）（P<0.05）.各试验组小鼠睾丸内DNA断裂的生殖细胞数量低于附睾内DNA断裂的精子数量.随着添加剂量的增加,小鼠睾丸内生殖细胞及附睾内精子凋亡率逐渐升高.表明小鼠生殖细胞凋亡及DNA损伤数量与镉剂量具有一定相关性.     

The dual specificity protein kinase CLK3 is abundantly expressed in mature mouse spermatozoa

CLK3, a member of the LAMMER family of dual-specificity protein kinases, is abundantly expressed in the reproductive system of male mice. Specifically, high levels of CLK3 protein expression are found in mature spermatozoa in the testis and epididymis. The majority of the CLK3 protein in the testis is a full-length kinase-containing form, and only a small amount of a catalytically inactive N-terminally truncated splice variant protein product is observed. Within the mature spermatozoa CLK3 is localized to the acrosome and tail. CLK3 is expelled from the sperm following the acrosome reaction and inactivated, likely by degradation by the proteases released by the sperm during the acrosome reaction. The CLK family of kinases has previously been implicated in mRNA splicing; however, the bulk of the CLK3 protein in these cells is located in the cytoplasm, suggesting that CLK3 may have additional roles in the cell.     

An amphioxus Msx gene expressed predominantly in the dorsal neural tube

 Genomic and cDNA clones of an Msx class homeobox gene were isolated from amphioxus (Branchiostoma floridae). The gene, AmphiMsx, is expressed in the neural plate from late gastrulation; in later embryos it is expressed in dorsal cells of the neural tube, excluding anterior and posterior regions, in an irregular reiterated pattern. There is transient expression in dorsal cells within somites, reminiscent of migrating neural crest cells of vertebrates. In larvae, mRNA is detected in two patches of anterior ectoderm proposed to be placodes. Evolutionary analyses show there is little phylogenetic information in Msx protein sequences; however, it is likely that duplication of Msx genes occurred in the vertebrate lineage. Received: 12 October 1998 / Accepted: 26 December 1998     

Cloning and expression of a novel human galectin cDNA, predominantly expressed in placenta(1)

A novel human galectin cDNA (PPL13) was isolated by screening a human 18-week fetal brain library. The mRNA was predominantly expressed in placenta, while the expression of it was not or barely detectable in heart, brain, lung, liver, skeletal muscle, kidney, and pancreas by Northern blot. COS-7 cells transfected with cDNA encoding human PPL13 sequestered the protein in nuclei although it lacked any known nuclear localization signal. STS of Unigene Hs. 24236 placed the cDNA to human chromosome 19q13.2.     

An abundant TIP expressed in mature highly vacuolated cells

Aquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS-PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So-deltaTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity-purified So-deltaTIP-specific peptide antibodies, we investigated the subcellular and tissue distribution of So-deltaTIP. So-deltaTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between delta-TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So-deltaTIP with that of more distantly related TIPs showed similarities with Arabidopsis gamma-TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So-deltaTIP may be induced when the large vacuole is formed.     

Immunolocalization of sperm protein 17 in human testis and ejaculated spermatozoa.

Sperm protein 17 (Sp17) is a highly conserved mammalian protein whose primary function is still poorly understood. Immunohistochemistry (IHC) in the human testis reveals the presence of Sp17 in some spermatocytes and abundantly in spermatids. All spermatogonia, Sertoli cells, and Leydig cells appear to be immunonegative for Sp17, whereas some interstitial cells are immunopositive. IHC recognized two distinct populations (immunopositive or not for Sp17) in the ejaculated spermatozoa. Although it will be necessary to clarify why some ejaculated spermatozoa do not contain Sp17, its distribution suggests that this protein may be associated with some phases of germinal cell differentiation.     

Genomic imprinting of IGF2, p57(KIP2) and PEG1/MEST in a marsupial, the tammar wallaby

Genomic imprinting is widespread amongst mammals, but has not yet been found in birds. To gain a broader understanding of the origin and significance of imprinting, we have characterized three genes, from three separate imprinted clusters in eutherian mammals in the developing fetus and placenta of an Australian marsupial, the tammar wallaby Macropus eugenii. Imprinted gene orthologues of human and mouse p57(KIP2), IGF2 and PEG1/MEST genes were isolated. p57(KIP2) did not show stable monoallelic expression suggesting that it is not imprinted in marsupials. In contrast, there was paternal-specific expression of IGF2 in almost all tissues, but the biased paternal expression of IGF2 in the fetal head and placenta, demonstrates the occurrence of tissue-specific imprinting, as occurs in mice and humans. There was also paternal-biased expression of PEG1/MESTalpha. The differentially methylated region (DMR) of the human and mouse PEG1/MEST promoter is absent in the wallaby. These data confirm the existence of common imprinted regions in eutherians and marsupials during development, but suggest that the regulatory mechanisms that control imprinted gene expression differ between these two groups of mammals.     

A novel spermatogenesis-specific uPAR gene expressed in human and mouse testis

The urokinase receptor, uPAR, which binds to the urinary-type plasminogen activator, controls matrix degradation in the processes of tissue remodeling, cell migration, and invasion. In the present study, we found a new urokinase receptor gene that encodes a 249-amino acid putative protein. Northern blot analysis showed specific expression in the testis of this gene, which we named the spermatogenesis-related gene (SGRG). In situ hybridization revealed a strong expression signal for SGRG in spermatogonia, but not in spermatocytes. Therefore, we conjecture that SGRG may regulate spermatocyte migration through breakdown of extracellular matrix protein barriers in spermatogenesis. Since SGRG is specifically expressed in spermatogonia, it provides an attractive candidate for development of a contraceptive vaccine.