Biomedical subjects

Yong-Lian Zhang

Publications and source records attributed to Yong-Lian Zhang.

At least 19 recordsLinked to original sources

PubMed2006

RNase9, an androgen-dependent member of the RNase A family, is specifically expressed in the rat epididymis.

Members of the RNase superfamily participate in a diverse array of biological processes, including RNA degradation, antipathogen activities, angiogenesis, and digestion. In the present study, we cloned the rat RNase9 gene by in silico methods and genome walking based on homology to the Macaca mulatta (rhesus monkey) epididymal RNase9. The gene is located on chromosome 15p14, spanning two exons, and is clustered with other members of the RNase A superfamily. It contains 1279 bp and encodes 182 amino acids, including a 24-amino acid signal peptide, and it has unique features known from other RNases. Unlike those other members, the rat RNase9 mRNA was specifically expressed in the epididymis, especially in the caput and corpus, and exhibited an androgen-dependent expression pattern but was downregulated in an epididymitis animal model. The RNASE9 was expressed in a principal cell-specific pattern. Interestingly, most of the principal cells in the caput expressed the RNASE9; however, in the distal caput, the principal cells showed a checkerboard-like pattern of immunoreactivity. We also observed that the RNASE9 was bound on the acrosomal domain of sperm. Its potential roles in sperm maturation are discussed.

Amino Acid Sequence↗

PubMed2006

Genome-wide profiling of segmental-regulated transcriptomes in human epididymis using oligo microarray.

Sperm maturation during passage through the epididymis depends on regionalized gene expression which maintains the progressively changing environment within the epididymal tubule. Towards defining the genes that drive the sequential maturation of spermatozoa, we profiled regionally regulated gene expression pattern in the epididymis of a fertile young male donor using Affymetrix human genome U133 plus 2.0 microarray representing approximately the whole human genome. Over 15000 transcripts, almost one-third of the total on the array were identified in whole epididymis. Among them, 65% were detected in all three regions of the epididymis, 410 or 2.6% were present only in one region and the remaining 32.4% were distributed in two regions. Region-specific transcripts observed in caput (264), corpus (61) and cauda (81) epididymides were further classified as empirically determined reported genes or ESTs. This study revealed for the first time, the expression in human epididymis of a number of region-specific genes. The original data will be made publicly available on the Shanghai Science and Technology Database (http://www.scbit.org/human_epididymis_transcriptomes).

Adult↗

PubMed2004

An epididymis-specific beta-defensin is important for the initiation of sperm maturation.

Although the role of the epididymis, a male accessory sex organ, in sperm maturation has been established for nearly four decades, the maturation process itself has not been linked to a specific molecule of epididymal origin. Here we show that Bin1b, a rat epididymis-specific beta-defensin with antimicrobial activity, can bind to the sperm head in different regions of the epididymis with varied binding patterns. In addition, Bin1b-expressing cells, either of epididymal origin or from a Bin1b-transfected cell line, can induce progressive sperm motility in immotile immature sperm. This induction of motility is mediated by the Bin1b-induced uptake of Ca(2+), a mechanism that has a less prominent role in maintaining motility in mature sperm. In vivo antisense experiments show that suppressed expression of Bin1b results in reduced binding of Bin1b to caput sperm and in considerable attenuation of sperm motility and progressive movement. Thus, beta-defensin is important for the acquisition of sperm motility and the initiation of sperm maturation.

Animals↗

PubMed2004

An effective method for raising antisera against beta-defensins: double-copy protein expression of mBin1b in E. coli.

Bin1b is a rat epididymis specific beta-defensin which may have fertility related functions in addition to its antimicrobial activity. beta-defensins are cysteine-rich cationic antimicrobial peptides that have their important implications in innate and adaptive immunity. Though considerable numbers of new beta-defensins have been discovered, few corresponding antibodies have been reported. The small peptide with special structure and antimicrobial nature of beta-defensins make them very difficult to express in prokaryotic system. Here we adopted a double-copy protein expression scheme based on which not only the mBin1b protein was successfully expressed but also the immunity of the antigen was enhanced. The validity of the antisera was verified by using Western blotting and immunohistochemical analyses. It will be a useful tool for deeply investigating the roles of Bin1b and also provide a simple but effective method in raising antisera against other members of the beta-defensin gene family.

Animals↗

PubMed2004

Spatial and temporal expression of germ cell nuclear factor in murine epididymis.

AIM: To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period. METHODS: The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope. RESULTS: GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis. The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis. In adults, GCNF exhibited a region-specific expression pattern, i.e., it was expressed predominantly in the initial segment, caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis. GCNF could be found in the nuclei of the principal, apical, narrow, clear and halo cells. CONCLUSION: GCNF may play an important role in epididymal differentiation and development and in sperm maturation.

Aging↗

PubMed2003

LCN6, a novel human epididymal lipocalin.

BACKGROUND: The lipocalin (LCN) family of structurally conserved hydrophobic ligand binding proteins is represented in all major taxonomic groups from prokaryotes to primates. The importance of lipocalins in reproduction and the similarity to known epididymal lipocalins prompted us to characterize the novel human epididymal LCN6. METHODS AND RESULTS: LCN6 cDNA was identified by database analysis in a comprehensive human library sequencing program. Macaca mulatta (rhesus monkey) cDNA was obtained from an epididymis cDNA library and is 93% homologous to the human. The gene is located on chromosome 9q34 adjacent LCN8 and LCN5. LCN6 amino acid sequence is most closely related to LCN5, but the LCN6 beta-barrel structure is best modeled on mouse major urinary protein 1, a pheromone binding protein. Northern blot analysis of RNAs isolated from 25 human tissues revealed predominant expression of a 1.0 kb mRNA in the epididymis. No other transcript was detected except for weak expression of a larger hybridizing mRNA in urinary bladder. Northern hybridization analysis of LCN6 mRNA expression in sham-operated, castrated and testosterone replaced rhesus monkeys suggests mRNA levels are little affected 6 days after castration. Immunohistochemical staining revealed that LCN6 protein is abundant in the caput epithelium and lumen. Immunofluorescent staining of human spermatozoa shows LCN6 located on the head and tail of spermatozoa with the highest concentration of LCN6 on the post-acrosomal region of the head, where it appeared aggregated into large patches. CONCLUSIONS: LCN6 is a novel lipocalin closely related to Lcn5 and Lcn8 and these three genes are likely products of gene duplication events that predate rodent-primate divergence. Predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility.

Amino Acid Sequence↗

PubMed2003

Androgen down-regulated and region-specific expression of germ cell nuclear factor in mouse epididymis.

Germ cell nuclear factor (GCNF), a nuclear orphan receptor, involved in spermatogenesis, neurogenesis, differentiation, and embryo development, was highly expressed with two transcripts (7.4 and 2.3 kb) in mouse testis and with only one transcript (7.4 kb) slightly expressed in brain, liver, and kidney. The 2.3-kb transcript was restricted to round spermatids at stages VII and VIII of the spermatogenic cycle. The present report demonstrated its expression in epididymis as well, but at a very low level. Northern blot analysis showed two transcripts: a common 7.4-kb transcript and a unique 3.1-kb transcript. The expression levels of both GCNF transcripts in epididymis were down-regulated by androgen, as observed in castrated animals and aged mice. Polyclonal antisera against GCNF protein were raised. Western blot analysis showed the presence of only one band in total protein extracts from either mouse testis or epididymis. It indicated that the two mRNAs (7.4 and 3.1 kb) encode for the same protein as in testis. Fluorescent immunohistochemical staining and in situ hybridization showed that its expression was in the principal cell abundant in the corpus region. It implies that some androgen-regulated gene expressions located at the corpus principal cells might be controlled by GCNF.

3' Untranslated Regions↗

PubMed2002

Analysis of germ cell nuclear factor transcripts and protein expression during spermatogenesis.

Germ cell nuclear factor (GCNF), an orphan receptor in the nuclear receptor superfamily, is expressed predominantly in developing germ cells in the adult mouse. Two Gcnf transcripts (7.4 and 2.1 kilobase [kb]) encoded by a single copy gene are expressed in the testis of several mammalian species. To identify features that regulate Gcnf expression, we characterized the structure and sequence of the mouse gene and its two transcripts and determined the expression profile of the GCNF protein during spermatogenesis. Genomic fragments spanning part of the 5'-untranslated region (UTR), the coding sequence, and the complete 3'-UTR (approximately 80 kb) were isolated and sequenced. The 3'-UTRs of the two transcripts are quite distinct. The 7.4 kb transcript, which appears earlier in spermatogenesis, has a very long 3'-UTR of 4451 nucleotides. In contrast, the 2.1 kb transcript, which is expressed predominantly during the haploid phase of spermatogenesis, has a 3'-UTR that is only 202 nucleotides in length. Additional analyses indicate that both transcripts share the same coding region and are associated with polysomes. A single GCNF protein band was detected in testis extracts by Western blotting with a specific antiserum. Immunohistochemical analysis showed that GCNF is localized in the nuclei of pachytene spermatocytes and round spermatids. GCNF is first detectable in early pachytene spermatocytes (stage II) and is continuously expressed until spermatids begin to elongate in stage IX. Although GCNF is generally distributed throughout the nucleus, it is particularly prominent in heterochromatic regions at some stages and in condensed chromosomes undergoing the meiotic divisions. This expression profile suggests that GCNF plays a role in transcriptional regulation during meiosis and the early haploid phase of spermatogenesis.

3' Untranslated Regions↗

PubMed2002

Cystatin 11: a new member of the cystatin type 2 family.

Cystatin (CST)11, a novel member of the CST type 2 family of cysteine protease inhibitors, was identified in Macaca mulatta epididymis by subtractive hybridization cloning. The human CST11 gene on chromosome 20p11.2 is located near three other CST genes expressed predominantly in the male reproductive tract. The CST11 gene spans three exons, a structure similar to that of other CST family 2 genes. An exon 2-deleted alternative transcript (CST11Delta2) was also identified. CST11 mRNA is expressed only in the epididymis as judged by Northern blot hybridization and is androgen regulated. The protein is most abundant in the initial segment, but is detected throughout the epididymis and on ejaculated human sperm. The calculated tertiary structure of CST11 reveals that the three regions corresponding to the protease inhibitory wedge of CST3 are similarly juxtaposed in CST11, consistent with protease inhibitor function. Intact and exon 2-deleted CST11 recombinant proteins were tested for antibacterial activity. After a 2-h incubation of Escherichia coli with 50 microg/ml recombinant CST11 or CST11Delta2, bacterial colony-forming units were reduced to 30% of control, indicating that both forms have antimicrobial activity.

Amino Acid Sequence↗

PubMed2002

[Expression, purification of the recombinant protein fragment of the monkey epididymis expressed gene ESc-615 and preparation of polyclonal antisera to it].

ESc-615 is a gene which was specifically expressed in epididymis, newly discovered in our laboratory. To elucidate the function of ESc-6 15 in sperm maturation and fertilization at the protein level, its C-terminal 310 amino ac id residues fragment was expressed in E.coli and used to immunize rabbits to obtain polyclonal antiserum with titer of 100 000. The antisera could detect th e antigen as low as 3 ng. A 63 kD protein in rat epididymis protein extracts could be detected in Western blot by using this particular antiserum, in accordance with the size of Esc-615 homologue polypeptide deduced from its cDNA. Immunostaining results showed that Esc-615 was a secreted protein and could bind sperm s, which was similar with other carboxylesterases in male reproductive system; t he positive staining was eliminated after antigen absorption. All these results showed that this antiserum was not only effective but also highly specific, providing a useful tool in further functional studies of Esc-615.

Animals↗

PubMed1998

A prostate-specific Protein Factor Inhibits the Interaction of Androgen Receptors with Hormone Response Elements.

The fragments of the androgen receptor (amino acids: 359-732) and of the glucocorticoid receptor (amino acids: 396-548) were expressed in E. coli as fusion proteins with GST. Both fusion proteins, denoted GST-AR and GST-GR, contained the DNA-binding domain and some flanking amino acids. In gel retardation assay both fusion proteins could bind the androgen/glucocorticoid response element (ARE/GRE). We found that both cytosol and nuclear extracts from rat ventral prostate (v.p), but not from other source tested could abolish the interaction of GST-AR and GST-GR with ARE/GRE (from C3 (1) gene and MMTV LTR). The inhibition was androgen-dependent and sensitive to temperature and trypsin treatment. It implies that a protein inhibitor was present in the rat ventral prostate.

Journal Article↗

PubMed1998

Expression of Fusion Protein Containing Androgen Receptor Segment and Preparation of Polyclonal Antibodies to Androgen Receptor.

The cDNA segment (1105-2224) of the androgen receptor was cloned into the pGEX-3X expression vector and expressed in E. coli. The soluble fusion proteins GST-AR were used to immunize rabbits to obtain polyclonal antibodies to androgen receptors. The antibodies were purified by GST-Sepharose 4B and indicated the specificity to androgen receptors. The purified antibodies can be used in study the function of AR.

Journal Article↗

PubMed1998

Nuclear Factors Enhance the Binding of Estrogen Receptor to Estrogen Response Element.

We obtained the full length of human estrogen receptor (hER) through the in vitro translation. It was shown that the translated product could bind to the estrogen response element (ERE). The nuclear extract prepared from the rat uterus after ovariectomy could enhance the binding of hER-ERE in an estrogen dependent manner. However, the enhancing effect was sharply decreased when the nuclear extract was pre-incubated at 50 degrees for 15 minutes before being used for the binding reaction. These results indicated the presence in the rat uterus extracts after ovariectomy of a heat labile factor that can enhance the binding of hER-ERE in an estrogen-dependent manner. The DNA binding domain of estrogen receptor (ER DBD) fused to the Scistosoma japonicam glutathione S-transferase (GST) was expressed in the E. coli. The expression product also could bind the ERE. However, the binding was not affected by the uterine extract, indicating that the heat-sensitive nuclear factors may interact with hER outside the DBD to enhance the binding of ER to ERE.

Journal Article↗

PubMed1998

Identification of the Subunits of C3P4 and Estimation of Their Molecular Weights by UV Cross-linking Assay.

The subunits and their molecular weights of an unknown DNA-binding protein C3P4 induced by androgen were studied by UV cross-linking assay. The results indicate that there are two subunits with molecular weights of about 110 kD and 27 kD in the C3P4 complex. Ferguson plot further shows the molecular weight of native C3P4 to be about 127 kD. It suggests that C3P4 very likely exists as heterodimer composed of one 110 kD subunit and one 27 kD subunit. The 110 kD subunit may play an important role in DNA-binding.

Journal Article↗

PubMed1998

Fluorescence-based Quantitative Analysis for mRNA in RT-PCR Process.

A DNA fragment that forms the template of a specific mRNA can be synthesized by reverse transcription PCR. In due number of cycle, the concentration of PCR product increases proportionally to that of the template cDNA, i.e., the corresponding mRNA in RT. Since the fluorescence quantum yield of ethidium bromide greatly increased when it was inserted into the DNA double helix, the extend of expression of mRNA can be calculated by determining the fluorescence intensity under selected excitation and emission wavelengths of PCR cDNA after a proper number of PCR cycle.

Journal Article↗

PubMed1998

Participation of Water in the Binding of Estrogen Receptor with Estrogen Responsive Element in vitro.

Many reports have showed that bound water was involved in the interaction between/among the macromolecules. However, it has not been reported whether bound water is also involved in the binding of trans-factors and cis-elements in the regulation of the eukaryotic gene trans-cription or not. Preliminary studies have been made on the effect of bound water on the binding of estrogen receptor with estrogen responsive element in vitro. In the gel retardation assay using the cytosol extract of rat uterus as the supplier of estrogen receptor and 32 bp oligonucleotide containing a concensus vitellogenin A(2) ERE as the probe, various cosolvents, such as glycerol, sucrose, N-dimethylformamide and dimethylsulfoxide, were added respectively to the reaction mixture in varying concentrations to regulate the osmotic pressure. The results indicated that the binding of ER-ERE was enhanced with the increase in the final concentration of these individual cosolvents. On the other hand, when the reaction was carried out under an increasing hydrostatic pressure, the ER-ERE binding was decreased sharply. After decompression the binding of ER-ERE was gradually restored to the normal level with the lapse of time. These results suggested that bound water was directly involved in the binding of ER-ERE and may play an important role in the regulation of the eukaryotic gene transcription.

Journal Article↗

PubMed1997

The Interaction of Recombinant Androgen Receptor with the Androgen Responsive Element.

A 1 119 bp (1105 to 2224) fragment of androgen receptor (AR) cDNA, named AR1 (containing the whole DNA binding domain, the hinge site region and the partial hormone binding domain) was constructed into an expression vector pGEX. The GST-ARI fusion protein was expressed in E. coli induced by IPTG and purified from glutathione-Sepharose-4B affinity column. With a known ARE of the C3(l) gene as positive probe, the expressed product was proved to be AR with high ARE-binding affinity by gel shifting assay(EMSA) and in vitro Dnase I footprinting analysis.

Journal Article↗

PubMed1997

Studies of the Expression of Progesterone Receptor Gene in the Rat Uterus during Early Pregnancy by RT-PCR.

Progesterone plays an important role in the establishment and maintenance of pregnancy. However, there have been no systematic reports on the expression of the rat uterine progesterone receptor (PR) gene during this process. In the present studies PR mRNA levels in rat uterus were analyzed during the estrous cycle and early pregnancy (d1-d10), using reverse transcription-polymerase chain reaction (RT-PCR). PR mRNA level was the highest at estrus and the lowest at metestrus, with the former value twice the latter. Post-coitum PR transcription increased rapidly, reached a peak of two fold of the d1 level on d3-d4 (before implantation), but drastically dropped to 50% of the d1 level on d6 (during implantation) and remained unchanged until d10. It is of interest to discover that the uterine PR mRNA level at the implantational sites was much higher than that at the non-implantational sites on d7-d10. Preliminary discussion was made on the physiological significance of these changes.

Journal Article↗