Publications by authors named "Tian-hua Huang"

40 Publications

Hepatitis B virus surface protein induces sperm dysfunction through the activation of a Bcl2/Bax signaling cascade triggering AIF/Endo G-mediated apoptosis.

Andrology 2021 05 1;9(3):944-955. Epub 2021 Feb 1.

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, China.

Background: Hepatitis B virus (HBV) was found to exist in semen and male germ cells of patients with chronic HBV infection. Our previous studies demonstrated that HBV surface protein (HBs) could induce sperm dysfunction by activating a calcium signaling cascade and triggering caspase-dependent apoptosis. However, the relationship between sperm dysfunction caused by HBs and caspase-independent apoptosis has not been investigated.

Objectives: To evaluate the effects of HBs exposure on sperm dysfunction by activating caspase-independent apoptosis.

Materials And Methods: Spermatozoa were exposed to HBs at concentrations of 0, 25, 50, and 100 μg/mL for 3 h. Flow cytometry, qRT-PCR, immunofluorescence assay, ELISA, and zona-free hamster oocyte penetration assays were performed.

Results: With increasing concentrations of HBs, various parameters of the spermatozoa changed. The number of Bcl2-positive cells declined and that of both Bax-positive cells and Apaf-1-positive cells increased. The transcription level of Bcl2 increased and that of both Bax and Apaf-1 declined. The average levels of AIF and Endo G declined in mitochondria and increased in the cytoplasm and nucleus. The sperm DNA fragmentation index increased. The mean percentages of live spermatozoa declined and that of both injured and dead spermatozoa increased; and the sperm penetration rate declined. For the aforementioned parameters, the differences between the test and the control groups were statistically significant.

Conclusion: HBs exposure can activate the Bax/Bcl2 signaling cascade that triggers AIF/Endo G-mediated apoptosis, resulting in sperm DNA fragmentation, sperm injury, and death, and a decrease in the sperm fertilizing capacity. This new knowledge will help to evaluate the negative impact of HBV on male fertility in HBV-infected patients.
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http://dx.doi.org/10.1111/andr.12965DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8247882PMC
May 2021

Transcription and regulation of hepatitis B virus genes in host sperm cells.

Asian J Androl 2018 May-Jun;20(3):284-289

Department of Genetics, Chengdu Jinxin Research Institute for Reproductive Medicine and Genetics, Chengdu 610066, China.

To investigate whether transcription of hepatitis B virus (HBV) gene occurs in human sperm, total RNA was extracted from sperm of patients with chronic HBV infection (test-1), from donor sperm transfected with a plasmid containing the full-length HBV genome (test-2), and from nontransfected donor sperm (control), used as the template for reverse transcription-polymerase chain reaction (RT-PCR). Positive bands for HBV DNA were observed in the test groups but not in the control. Next, to identify the role of host genes in regulating viral gene transcription in sperm, total RNA was extracted from 2-cell embryos derived from hamster oocytes fertilized in vitro by HBV-transfected (test) or nontransfected (control) human sperm and successively subjected to SMART-PCR, suppression subtractive hybridization, T/A cloning, bacterial amplification, microarray hybridization, sequencing and the Basic Local Alignment Search Tool (BLAST) search to isolate differentially expressed genes. Twenty-nine sequences showing significant identity to five human gene families were identified, with chorionic somatomammotropin hormone 2 (CSH2), eukaryotic translation initiation factor 4 gamma 2 (EIF4G2), pterin-4 alpha-carbinolamine dehydratase 2 (PCBD2), pregnancy-specific beta-1-glycoprotein 4 (PSG4) and titin (TTN) selected to represent target genes. Using real-time quantitative RT-PCR (qRT-PCR), when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNA interference, transcriptional levels of HBV s and x genes significantly decreased (or increased) (P < 0.05). Silencing of a control gene in sperm did not significantly change transcription of HBV s and x genes (P > 0.05). This study provides the first experimental evidence that transcription of HBV genes occurs in human sperm and is regulated by host genes.
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http://dx.doi.org/10.4103/aja.aja_46_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5952484PMC
June 2019

Host genes regulate transcription of sperm-introduced hepatitis B virus genes in embryo.

Reprod Toxicol 2017 10 16;73:158-166. Epub 2017 Aug 16.

Jinxin Research Institute for Reproductive Medicine and Genetics, Chengdu Jinjiang Hospital for Maternal and Child Health Care, 66 Jinxiu Road, Chengdu 610066, China; Research Center for Reproductive Medicine, Shantou University Medical College, 22 Xinling Road, Shantou 515041, China. Electronic address:

Hepatitis B virus (HBV) can invade the male germline, and sperm-introduced HBV genes could be transcribed in embryo. This study was to explore whether viral gene transcription is regulated by host genes. Embryos were produced by in vitro fertilization of hamster oocytes with human sperm containing the HBV genome. Total RNA extracted from test and control embryos were subjected to SMART-PCR, SSH, microarray hybridization, sequencing and BLAST analysis. Twenty-nine sequences showing significant identity to five human gene families were identified, with CSH2, EIF4G2, PCBD2, PSG4 and TTN selected to represent target genes. Using qRT-PCR, when CSH2 and PCBD2 (or EIF4G2, PSG4 and TTN) were silenced by RNAi, transcriptional levels of HBV s and x genes decreased (or increased). This is the first report that host genes participate in regulation of sperm-introduced HBV gene transcription in embryo, which is critical to prevent negative impact of HBV infection on early embryonic development.
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http://dx.doi.org/10.1016/j.reprotox.2017.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7127588PMC
October 2017

G protein-coupled estrogen receptor-protein kinase A-ERK-CREB signaling pathway is involved in the regulation of mouse gubernaculum testis cells by diethylstilbestrol.

Arch Environ Contam Toxicol 2014 Jul 4;67(1):97-103. Epub 2013 Dec 4.

Department of Pediatric Surgery, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, China.

The etiology of testicular dysgenesis syndrome is multifactorial and involves environmental factors, such as environmental estrogens. Several studies have shown that hormonal effects on the gubernaculum may affect testicular descent. Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen that disrupts the morphology and proliferation of gubernacular cells, but the underlying mechanisms remain elusive. In this study, we aimed to determine whether DES may regulate the function of gubernaculum testis cells by way of nongenomic effects mediated by G protein-coupled estrogen receptor (GPER). We used cultured mouse gubernacular testis cells to demonstrate that GPER is expressed in gubernaculum testis cells. Erk1/2 inhibitor PD98059, PKA inhibitor H89, and Src inhibitor PP2 relieved DES-induced inhibition of gubernaculum testis cell proliferation, but ER inhibitor ICI 182780 had no effects on DES-induced inhibition of gubernaculum testis cell proliferation. In addition, we found that DES induced the activation of CREB downstream of PKA, Src, and ERK1/2 in these cells. These data suggest that the effects of DES on mouse gubernaculum testis cells are mediated at least partially by GPER-protein kinase A-ERK-CREB signaling pathway.
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http://dx.doi.org/10.1007/s00244-013-9976-3DOI Listing
July 2014

Diethylstilbestrol impairs the morphology and function of mouse gubernaculum testis in culture.

Cell Biol Toxicol 2012 Dec 4;28(6):397-407. Epub 2012 Sep 4.

Department of Pediatric Surgery, The Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong, People's Republic of China.

Diethylstilbestrol (DES) is a nonsteroidal synthetic estrogen widely used in estrogen therapy. In animal models, exposure to DES disrupts the outgrowth of the gubernacula, leading to testis maldescent. However, it remains unclear whether the effects of DES on gubernaculum are direct or indirect, and the underlying mechanisms are largely obscure. In this study, mouse gubernaculum testis cells were isolated and treated by DES, and cell morphology and function were examined. The results showed that DES changed the morphology and inhibited the proliferation of gubernacular cells. Furthermore, DES increased intracellular [Ca(2+)] and induced F-actin rearrangement and stress fiber formation in gubernaculum testis cells in a dose-dependent manner. Taken together, these data suggest that DES impairs the morphology and inhibits the proliferation and contractility of gubernaculum testis cells. The experimental model we established and our observations based on this model help provide new insight into the role of DES in the etiology of cryptorchidism.
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http://dx.doi.org/10.1007/s10565-012-9231-0DOI Listing
December 2012

The integrated HIV-1 provirus in patient sperm chromosome and its transfer into the early embryo by fertilization.

PLoS One 2011 14;6(12):e28586. Epub 2011 Dec 14.

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, China.

Complete understanding of the route of HIV-1 transmission is an important prerequisite for curbing the HIV/AIDS pandemic. So far, the known routes of HIV-1 transmission include sexual contact, needle sharing, puncture, transfusion and mother-to-child transmission. Whether HIV can be vertically transmitted from human sperm to embryo by fertilization is largely undetermined. Direct research on embryo derived from infected human sperm and healthy human ova have been difficult because of ethical issues and problems in the collection of ova. However, the use of inter-specific in vitro fertilization (IVF) between human sperm and hamster ova can avoid both of these problems. Combined with molecular, cytogenetical and immunological techniques such as the preparation of human sperm chromosomes, fluorescent in situ hybridization (FISH), and immunofluorescence assay (IFA), this study mainly explored whether any integrated HIV provirus were present in the chromosomes of infected patients' sperm, and whether that provirus could be transferred into early embryos by fertilization and maintain its function of replication and expression. Evidence showed that HIV-1 nucleic acid was present in the spermatozoa of HIV/AIDS patients, that HIV-1 provirus is present on the patient sperm chromosome, that the integrated provirus could be transferred into early embryo chromosomally integrated by fertilization, and that it could replicate alongside the embryonic genome and subsequently express its protein in the embryo. These findings indicate the possibility of vertical transmission of HIV-1 from the sperm genome to the embryonic genome by fertilization. This study also offers a platform for the research into this new mode of transmission for other viruses, especially sexually transmitted viruses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028586PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237474PMC
August 2012

Human umbilical cord mesenchymal stem cells derived from Wharton's jelly differentiate into insulin-producing cells in vitro.

Chin Med J (Engl) 2011 May;124(10):1534-9

Department of Pediatrics, Second Affiliated Hospital of Shantou University Medical College, Shantou, Guangdong 515041, China.

Background: Islet transplantation is an effective way of reversing type I diabetes. However, islet transplantation is hampered by issues such as immune rejection and shortage of donor islets. Mesenchymal stem cells can differentiate into insulin-producing cells. However, the potential of human umbilical cord mesenchymal stem cells (huMSCs) to become insulin-producing cells remains undetermined.

Methods: We isolated and induced cultured huMSCs under islet cell culture conditions. The response of huMSCs were monitored under an inverted phase contrast microscope. Immunocytochemical and immunofluorescence staining methods were used to measure insulin and glucagon protein levels. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to detect gene expression of human insulin and PDX-1. Dithizone-staining was employed to determine the zinc contents in huMSCs. Insulin secretion was also evaluated through radioimmunoassay.

Results: HuMSCs induced by nicotinamide and β-mercaptoethanol or by neurogenic differentiation 1 gene (NeuroD1) transfection gradually changed morphology from typically elongated fibroblast-shaped cells to round cells. They had a tendency to form clusters. Immunocytochemical studies showed positive expression of human insulin and glucagon in these cells in response to induction. RT-PCR experiments found that huMSCs expressed insulin and PDX-1 genes following induction and dithizone stained the cytoplasm of huMSCs a brownish red color after induction. Insulin secretion in induced huMSCs was significantly elevated compared with the control group (t = 6.183, P < 0.05).

Conclusions: HuMSCs are able to differentiate into insulin-producing cells in vitro. The potential use of huMSCs in β cell replacement therapy of diabetes needs to be studied further.
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May 2011

Several types of soft tissue sarcomas originate from the malignant transformation of adipose tissue-derived stem cells.

Mol Med Rep 2010 May-Jun;3(3):441-8

Central Laboratory, Chaozhou Central Hospital, Chaozhou 521021, P.R. China.

The cellular origin of soft tissue sarcomas (STSs) is not fully understood. The cancer stem cell hypothesis presumes that tumors originate from the malignant transformation of stem cells. As a type of multipotent stem cell, adipose tissue-derived stromal/stem cells (ADSCs), which possess an unexpected degree of plasticity and often reside in other tissues, may represent a potential source of soft tissue sarcoma. To ascertain whether ADSCs are responsible for the formation of STSs, ADSCs from mice were cultured and treated with 3-methycholanthrene to derive transformed cells. These transformed ADSCs were then injected subcutaneously into immunodeficient mice to test their tumorigenic potential. We found that they generated several types of STSs, including synovial sarcoma, malignant fibrous histiocytoma and fibrosarcoma. This is the first study to report that ADSCs may be the potential initiating cells for synovial sarcoma. Our findings indicate that STSs might originate from malignantly transformed ADSCs.
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http://dx.doi.org/10.3892/mmr_00000277DOI Listing
October 2012

In vitro study on vertical transmission of the HIV-1 gag gene by human sperm.

J Med Virol 2011 Jan;83(1):16-23

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, China.

HIV/AIDS is a major public health problem worldwide. To explore the feasibility of HIV vertical transmission by human sperm, plasmid construction and transfection, interspecific in vitro fertilization of zona-free hamster ova by human sperm, fluorescence in situ hybridization (FISH), RT-PCR, and immunofluorescence assay (IFA) were carried out. The FISH signals for HIV-1 gag DNA were observed in the nuclei and chromosomes of transfected human sperm, male pronuclei of zygotes, and nuclei of blastomeres of two-cell embryos, indicating that the HIV-1 gag gene could be transmitted via the sperm membrane and integrated into the sperm genome. In contrast, human sperm carrying the target gene achieved normal fertilization, and replication of the sperm-mediated target gene was synchronized with the host genome. Using RT-PCR, the positive bands for the target gene were observed in the transfected human sperm and two-cell embryos. These results further confirm that the target gene can be transcribed into mRNA in human sperm and embryonic cells. Positive signals for the HIV-1 p24 gag protein were shown by IFA in two-cell embryos containing the sperm-mediated target gene and not in the transfected human sperm, which indicated that the sperm-mediated target gene could be translated to make HIV-1 p24 gag protein in embryonic cells, but not in sperm cells. The results provide evidence for possible vertical transmission of the HIV-1 gag gene to the embryo by fertilizing sperm in vitro.
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http://dx.doi.org/10.1002/jmv.21767DOI Listing
January 2011

[Effects of diethylstilbestrol on cultured testis gubernacular cells in mice].

Zhonghua Nan Ke Xue 2009 Oct;15(10):872-5

Department of Pediatric Surgery, The Second Hospital, China.

Objective: To establish a primary culture of the testis gubernacular cells of Kunming mice, observe the morphological characteristics of the cells, and explore the effects of exogenous estrogens (EEs) on the development of the testis gubernacula in vitro.

Methods: We removed the gubernacula from 3-day-old mice with the surgical magnifier and cultured the gubernacular cells. Then we detected the cell viability by trypan blue and cell morphology by HE staining. The subcultured cells were randomly divided into a blank control, a DMSO (0.1%, v/v) control, and 4 experimental groups (given 0.01, 0.10, 1.00 and 10.00 micdrog/ml of diethylstilbestrol [DES] dissolved in DMSO, respectively). After treated for 12, 24 and 48 hours, the gubernacular cells were observed for morphological changes and proliferation inhibition by CCK-8.

Results: Most of the cultured gubernacular cells were fibroblasts, and a few were epithelioids. The primary cells showed a viability of 85%-90%. Dose- and time-dependent inhibition of cell proliferation was found in the four experimental groups at three different times, with statistically significant differences (P < 0.01).

Conclusion: Gubernacular cells can be cultured in vitro. EEs inhibit the proliferation of gubernacular cells in a dose- and time-dependent manner. An in- sight into the effects EES on cultured gubernacular cells is an effective approach to the study of their influence on the development of the reproductive system.
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October 2009

Differentiation of human umbilical cord Wharton's jelly-derived mesenchymal stem cells into germ-like cells in vitro.

J Cell Biochem 2010 Mar;109(4):747-54

Department of Pediatrics, Second Affiliated Hospital of Shantou University Medical College (SUMC), Shantou, PR China.

Recent studies have demonstrated that mesenchymal stem cells could differentiate into germ cells under appropriate conditions. We sought to determine whether human umbilical cord Wharton's jelly-derived mesenchymal stem cells (HUMSCs) could form germ cells in vitro. HUMSCs were induced to differentiate into germ cells in all-trans retinoic acid, testosterone and testicular-cell-conditioned medium prepared from newborn male mouse testes. HUMSCs formed "tadpole-like" cells after induction with different reagents and showed both mRNA and protein expression of germ-cell-specific markers Oct4 (POUF5), Ckit, CD49(f) (alpha6), Stella (DDPA3), and Vasa (DDX4). Our results may provide a new route for reproductive therapy involving HUMSCs and a novel in vitro model to investigate the molecular mechanisms that regulate the development of the mammalian germ lineage.
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http://dx.doi.org/10.1002/jcb.22453DOI Listing
March 2010

In vivo study on vertical transmission of the HIV-1 gag gene via mouse oocytes.

Curr HIV Res 2009 Sep;7(5):562-8

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, Guangdong, 515041, PR China.

Acquired immunodeficiency syndrome (AIDS) is a major public health problem worldwide. This study was performed to explore the feasibility of vertical transmission of human immunodeficiency virus-1 (HIV-1) gag gene via oocyte. The recombinant plasmid (pIRES2-EGFP-gag) was injected into mouse ovaries to transfect germ cells. Induction of superovulation and then animal mating were performed to collect oocytes and two-cell embryos. Positive FISH signals for HIV-1 gag DNA were detected in the nuclei of oocytes and embryos, and in chromosomes of mature oocytes, indicated integration of the gene into the oocyte genome and gene replication in the embryo. HIV-1 gag cDNA positive bands detected by RT-PCR in oocytes and embryos indicated successful gene transcription, while positive immunofluorescence signals for HIV-1 gag protein indicated successful translation in both oocytes and embryos. The HIV-1 gag gene was transmitted vertically to the next generation via oocytes and it retained its function in replication, transcription and translation following at least one mitotic division in embryos.
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http://dx.doi.org/10.2174/157016209789346237DOI Listing
September 2009

FT-IR methodology for quality control of arabinogalactan protein (AGP) extracted from green tea (Camellia sinensis ).

J Agric Food Chem 2009 Jun;57(12):5121-8

Shantou University Medical College, Shantou, Guangdong, China.

A rapid methodology of quality control was developed for arabinogalactan proteins (AGP) extracted and purified from green tea. Using the vectorial angle method and IR spectrum analysis, the 1200-800 cm(-1) region in second-derivative IR spectra was determined as the key fingerprinting region of green tea AGP, with the 1090-900 cm(-1) region reflecting their conservative and common characteristics. In fact, the key monosaccharides, galactose (Gal) and arabinose (Ara), were shown to have intense peaks at about 1075 and 1045 cm(-1), respectively, and uronic acids at about 1018 cm(-1) in second-derivative IR spectra. The variable region was identified to be at about 1134-1094 and 900-819 cm(-1) and was probably due to compositional and structural differences between AGPs. The constructed methodology was tested on green tea AGP extracted by three treatments and purified to apparent homogeneity as water-extracted Camellia sinensis AGP (CSW-AGP), pectinase-extracted C. sinensis AGP (CSP-AGP), and trypsin-extracted C. sinensis AGP (CST-AGP) with an Ara/Gal ratio of 1.37, 1.57, and 1.82, respectively. Regarding in vitro antioxidant activity, the AGPs (CSW-AGP and CST-AGP) with higher similarity (closer cos theta values calculated for second-derivative IR spectra) exhibited a similar ability of chelating ferrous ions and had a similar capability for scavenging hydroxyl radicals. In conclusion, the combination of second-derivative IR spectrum analysis and the vectorial angle method has allowed a successful characterization of green tea AGPs and was shown to be suitable for their compositional and activity discrimination and rapid quality evaluation.
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http://dx.doi.org/10.1021/jf803707aDOI Listing
June 2009

[Key sperm membrane proteins in sperm-egg fusion].

Zhonghua Nan Ke Xue 2009 Mar;15(3):261-4

Research Center for Reproductive Medicine, Department of Cell Biology and Genetics, Medical College of Shantou University, Shantou, Guangdong 515041, China.

Fertilization is a complex process involving multiple steps, of which sperm-egg fusion is most important. This article presents a detailed review of some of the key sperm membrane proteins closely related with fertilization, such as the Izumo, the ADAMs gene family and the Crisp gene family proteins, which is of practical significance for deeper insights into the mechanisms of sperm-egg fusion, as well as for the improvement of clinical diagnosis of male infertility and development of novel contraceptive drugs.
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March 2009

Effects of hepatitis B virus S protein on human sperm function.

Hum Reprod 2009 Jul 11;24(7):1575-83. Epub 2009 Mar 11.

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou 515041, People's Republic of China.

Background: Hepatitis B virus (HBV) has been determined to exist in semen and male germ cells from patients with chronic HBV infection, but no data are yet available on the impact of HBV S protein (HBs), the main component of HBV envelop protein, on the human reproductive system. The purpose of this article was to investigate the effect of HBs on human sperm function.

Methods: Sperm motility analyses, sperm penetration assays, mitochondrial membrane potential assays, immunolocalizations with confocal microscopy and flow cytometry analyses were performed.

Results: HBs reduced sperm motility in a dose- and time-dependent manner and caused the loss of sperm mitochondrial membrane potential. HBs-HBs monoclonal antibody (MAb) complex apparently aggravated such impairments. After 4 h incubation with HBs at concentrations of 25, 50, 100 microg/ml, the percentages of sperm motility a+b significantly decreased compared with the control (P < 0.01). The fertilization rate and the fertilizing index in HBs-treated group were 40% and 0.57, respectively, which were significantly lower than 90% and 1.6, respectively, in the control (P < 0.01). The asialoglycoprotein receptor (ASGP-R) and HBs were found to localize mainly on the postacrosomal region. Both ASGP-R MAb and asialofoetuin, a high-affinity ligand of ASGP-R, inhibited the HBs-caused loss of sperm motility and mitochondrial membrane potential.

Conclusions: HBs had adverse effects on human sperm function, and ASGP-R may play a role in the uptake of HBs into sperm cells, as demonstrated by the competitive inhibition of ASGP-R MAb or asialofoetuin, resulting in diminished impairment caused by HBs.
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http://dx.doi.org/10.1093/humrep/dep050DOI Listing
July 2009

In vitro and in vivo studies evaluating recombinant plasmid pCXN2-mIzumo as a potential immunocontraceptive antigen.

Am J Reprod Immunol 2009 Mar;61(3):227-35

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, Guangdong, China.

Problems: Study on feasibility of pCXN2-mIzumo as a potential immunocontraceptive antigen.

Method Of Study: Two groups of mice received 100 microg/mouse plasmids of pCXN2-mIzumo and pCXN2 respectively. RT-PCR Immunofluorescence assay and ELISA were performed to observe pCXN2-mIzumo expression and antibody response in the inoculated mice. Sperm penetration assay and animal mating were employed to detect differences of in vitro fertilization (IVF) rate and mean litter size between the experimental and control groups.

Results: Izumo cDNA positive bands were detected in sample from mice immunized with pCXN2-mIzumo. IgG response started to rise at 2 weeks after first boost and reached the highest antibody titers at 2 weeks after third boost of immunization with pCXN2-mIzumo in the experimental mice. In vitro fertilization rate in the experimental group (11.57%) was significantly lower than that in control (36.60%). Significant difference of mean litter size between female experimental and control groups was observed, and there was significant negative correlation between individual anti-serum titers and litter size (r = -0.308, P < 0.05).

Conclusion: pCXN2-mIzumo plasmid possesses appreciable anti-fertility potential.
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http://dx.doi.org/10.1111/j.1600-0897.2009.00685.xDOI Listing
March 2009

In vitro and in vivo studies evaluating antisemen antibodies as a potential spermicidal agent in hamsters.

Fertil Steril 2009 Sep 4;92(3):1116-1123. Epub 2008 Oct 4.

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, Guangdong, People's Republic of China 515041. Electronic address:

Objective: To determine the spermicidal activity of antisemen antibodies in the hamster model.

Design: Prospective, controlled study.

Setting: Advanced preclinical sciences center.

Animal(s): Subgroups of 10 and 14 golden hamsters.

Intervention(s): Ex vitro and in vivo treatment of sperm with antisemen antibodies or normal rabbit serum.

Main Outcome Measure(s): The EC(50) value of antisemen antibodies, the time required for 50% motility loss of progressively motile spermatozoa exposed to antisemen antibodies, the average sperm mitochondrion fluorescence intensity, the rate of fertilization, and the scoring of histologic changes in the hamster vaginal tissue.

Result(s): The EC(50) value of antisemen antibodies was found 70 microg/mL, and the time required for 50% motility loss of progressively motile spermatozoa exposed to antisemen antibodies (at 70 microg/mL) was 5 minutes; for the experimental and control groups, the average fluorescence intensities of sperm mitochondria were respectively 180.28 +/- 82.24 and 309.74 +/- 148.37, the fertilization rates in vitro were 0.09% and 45%, the rates of fertilization with intrauterine sperm injection were 0 and 15.0%. There was a significant difference between two groups. None of the four hamsters that received antisemen antibodies in gel-polyoxyl-40-stearate had epithelial disruption characteristic of inflammation.

Conclusion(s): Antisemen antibodies possess appreciable spermicidal potential, which may be explored as an effective constituent of spermicide.
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http://dx.doi.org/10.1016/j.fertnstert.2008.07.1748DOI Listing
September 2009

A sensitive and rapid assay for investigating vertical transmission of hepatitis B virus via male germ line using EGFP Vector as reporter.

J Biomed Biotechnol 2008 ;2008:495436

Research Center for Reproductive Medicine, Medical College, Shantou University, Shantou 515041, China.

Hepatitis B virus (HBV) constitutes a serious menace to man. DNA recombination and sequencing, interspecific in vitro fertilization, single-embryo PCR and RT-PCR were employed to establish a sensitive and rapid assay for exploring the vertical transmission of viruses via male germ line. Plasmid pIRES2-EGFP-HBs which expressed enhanced green fluorescent protein as reporter for the expression of hepatitis B virus S gene was successfully constructed and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize with zona-free hamster ova. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBs DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive control (PCR) indicating expression of pIRES2-EGFP-HBs, and not observed in the embryos without green fluorescence and negative controls (PCR and RT-PCR) indicating no pIRES2-EGFP-HBs in the cells. The advantages and application foreground of this assay for study on vertical transmission of viruses such as HCV, HIV, HPV, and SARS via germ line were discussed.
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http://dx.doi.org/10.1155/2008/495436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2486355PMC
September 2008

Single-chain antibody against human lipocalin-type prostaglandin D synthase: construction, expression, purification, and activity assay.

Biochemistry (Mosc) 2008 Jun;73(6):702-10

Research Center for Reproductive Medicine, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong 515041, China.

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.
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http://dx.doi.org/10.1134/s0006297908060114DOI Listing
June 2008

Cytogenetic analysis of in vivo and in vitro matured oocytes derived from naturally cycling and stimulated mice.

Syst Biol Reprod Med 2008 May-Jun;54(3):155-62

The First Affiliated Hospital of Shantou University Medical College, Shantou, China.

The objective of the study was to analyze the potential role of follicle stimulating hormone (FSH) in cytogenetic changes of in vivo and in vitro matured mouse oocytes and to determine whether the lower developmental potential of immature oocytes is due to a higher incidence of abnormalities in meiotic spindle organization and chromosome alignment as well as aneuploidy. In vivo matured oocytes were collected from naturally ovulated and superovulated (5.0 I U of recombinant follicle-stimulating hormone [rec-FSH] + recombinant human chorionic gonadotropin [rec-HCG]) mice. Immature oocytes were retrieved from naturally cycling mice and from mice primed with rec-FSH for 48 h. The immature oocytes were cultured 18 h for in vitro maturation (IVM). In vivo and in vitro matured oocytes were assessed for the meiotic spindle organization and chromosome alignment as well as aneuploidy. There was no significant difference of meiotic spindle organization, chromosomal alignment and aneuploidy between in vivo and in vitro matured oocytes derived from naturally cycling and stimulated mice. Therefore, the lower developmental potential of immature oocytes does not appear to be directly related to the incidence of abnormal meiotic spindle organization and chromosome alignment or to aneuploidy.
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http://dx.doi.org/10.1080/19396360802102012DOI Listing
September 2008

An improved experimental model for studying vertical transmission of hepatitis B virus via human spermatozoa.

J Virol Methods 2008 Jul 23;151(1):116-20. Epub 2008 Apr 23.

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou 515041, China.

Study of the mechanism of transmission of hepatitis B virus is important for public health. An improved experimental model is described for studying vertical transmission of hepatitis B virus (HBV) via human spermatozoa. Recombinant plasmid pIRES2-EGFP-HBx which would express enhanced green fluorescent protein (EGFP) used as a marker for the expression of hepatitis B virus X (HBx) gene was constructed successfully and confirmed by PCR, EcoR I and Sal I digestion, and DNA sequencing. After exposure to the plasmid, human spermatozoa were used to fertilize zona-free hamster ova in vitro. Two-cell embryos were collected and classified into group A with green fluorescence and group B without green fluorescence under fluorescence microscope. The results showed that HBx DNA positive bands were detected in the embryos with green fluorescence (PCR and RT-PCR) and positive controls (PCR) indicating presence and expression of HBx gene. In contrast, HBx gene expression was not detected in the embryos without green fluorescence and negative controls (PCR and RT-PCR). This improved experimental model is more efficient, accurate and reliable for studying further perinatal transmission of HBV or other viruses causing chronic human disease possibly via the male germ line.
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http://dx.doi.org/10.1016/j.jviromet.2008.03.014DOI Listing
July 2008

Investigation of recombinant mouse sperm protein izumo as a potential immunocontraceptive antigen.

Am J Reprod Immunol 2008 Mar;59(3):225-34

Research Center for Reproductive Medicine, Shantou University Medical College, Shantou, Guangdong, China.

Problem: To determine if the recombinant mouse Izumo (mIzumo) could be used as a potential immunocontraceptive antigen.

Method Of Study: The recombinant mIzumo fused with 6His tag (6His-mIzumo) was purified by immobilized Ni2+ affinity chromatography. Enzyme-linked immunosorbent assay and Western blot were used to detect anti-6His-mIzumo activities of serum from the mice immunized with 6His-mIzumo. Inhibition of the anti-6His-mIzumo antibody on mouse sperm-egg fusion in vitro was performed using the zona free oocytes and acrosome reacted sperm. Fertility of the 6His-mIzumo immunized male and female mice was compared with control mice.

Results: The recombinant mIzumo was successfully produced. Female and male mice inoculated with 6His-mIzumo developed a specific serum antibody and the highest antibody titer lasted at least 6 weeks. The serum anti-6His-mIzumo antibody almost completely blocked mouse sperm-egg fusion in vitro. However, there was no significant reduction in fertility for both male and female mice immunized with 6His-mIzumo compared with control mice.

Conclusion: The circulated anti-mIzumo antibody can block mouse sperm-egg fusion in vitro but has no effect on fertility in vivo. It seems that application of Izumo as a candidate antigen in development of contraceptive vaccine needs further investigation.
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http://dx.doi.org/10.1111/j.1600-0897.2007.00571.xDOI Listing
March 2008

Combined effects of radiotherapy and endostatin gene therapy in melanoma tumor model.

Radiat Environ Biophys 2008 Apr 30;47(2):285-91. Epub 2007 Nov 30.

Research Center for Reproductive Medicine, Shantou University Medical College, 515041, Shantou, China,

PEgr-Endostatin-EGFP plasmid was constructed to investigate its expression properties induced by ionizing irradiation and the effect of pEgr-Endostatin-EGFP gene-radiotherapy on melanoma tumor-bearing mice. The pEgr-Endostatin-EGFP plasmid was transfected into B16 cell line with liposome. The expression property of endostatin was investigated by RT-PCR and that of EGFP was detected by flow cytometry. Tumor-bearing mice were treated by the plasmid injection and 2 Gy X-irradiation of three fractions. Tumor growth was observed for 18 days after treatment. Change of tumor capillary formation was measured with histochemistry assay at the end of the experiment. The expression of GFP in B16 melanoma cells was detected after X-irradiation with 0.05-20 Gy. Time-course studies showed that the expression of GFP in B16 cells reached its peak at 8 h after irradiation with 2 Gy. The injection of pEgr-Endostatin-EGFP recombinant plasmid into the implanted B16 melanoma in C57BL/6J mice followed by local X-irradiation could significantly inhibit tumor growth with inhibition of intratumor micro-vessel density. The inhibitory effect of pEgr-Endostatin-EGFP gene-radiotherapy on the growth of B16 melanoma is correlated with the marked decrease of intratumoral vascularization. The present data point to the potential of an anti-angiogenic approach in gene-radiotherapy of cancer.
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http://dx.doi.org/10.1007/s00411-007-0144-xDOI Listing
April 2008

Effect of polyvinylpyrrolidone on bovine oocyte maturation in vitro and subsequent fertilization and embryonic development.

Reprod Biomed Online 2007 Aug;15(2):198-207

Department of Obstetrics and Gynecology, McGill University, Montreal, Canada H3A 1A1.

The exact role of polyvinylpyrrolidone (PVP) in culture medium for oocyte maturation is still largely unknown. Bovine cumulus-oocyte complexes (COC) were cultured in in-vitro maturation (IVM) medium supplemented with 10% fetal bovine serum (FBS), 0.3% PVP (K-90) or 10% serum substitute supplement (SSS) respectively. The rates of oocyte maturation, fertilization and early embryonic development were evaluated. In addition, the status of DNA fragmentation in the oocytes was determined by comet assay, and the ratio of trophectoderm (TE) cells and inner cell mass (ICM) in blastocysts was determined by differential staining. Furthermore, the percentage of apoptotic cells in the blastocysts was examined by TUNEL assay. The results indicated that the effect of PVP in IVM medium was similar to FBS in terms of oocyte maturation and subsequent embryonic development. However, the addition of SSS in IVM medium retarded further embryonic development and resulted in more oocyte DNA fragmentation and a higher ratio of TE cells and ICM in the blastocysts. However, the number of apoptotic cells in blastocysts was similar among the three groups. These results suggest for the first time that the addition of PVP in oocyte maturation medium is not only a suitable substitute for serum but is also beneficial to in-vitro oocyte maturation.
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http://dx.doi.org/10.1016/s1472-6483(10)60709-2DOI Listing
August 2007

Relationship between contents of lipocalin-type prostaglandin D synthase on the surface of infertility sperm and in seminal plasma.

Biochemistry (Mosc) 2007 Feb;72(2):215-8

Research Center for Reproductive Medicine, Department of Cell Biology and Genetics, Shantou University Medical College, Shantou, Guangdong, China.

Lipocalin-type prostaglandin D synthase (L-PGDS) is localized in Leydig cells, sperm, and epithelial cells of the epididymis. The present study was to determine the correlation between content of this enzyme in seminal plasma and on the surface of sperm. We analyzed 90 semen samples. L-PGDS in seminal plasma was analyzed by an ELISA procedure. L-PGDS on sperm was analyzed by flow cytometry. The semen donors were categorized in three groups: normal, oligospermic, and azoospermic. According to results obtained, L-PGDS may have the ability to improve progressive motility of sperm, and L-PGDS in seminal plasma and on sperm surface may impact male fertility in the female reproductive tract.
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http://dx.doi.org/10.1134/s0006297907020125DOI Listing
February 2007

Bone marrow stromal cells express neural phenotypes in vitro and migrate in brain after transplantation in vivo.

Biomed Environ Sci 2006 Oct;19(5):329-35

Department of Biology, Medical College of Shantou University, Shantou 515031, Guangdong, China.

Objective: To investigate the differentiation of bone marrow stromal cells (BMSC) into neuron-like cells and to explore their potential use for neural transplantation.

Methods: BMSC from rats and adult humans were cultured in serum-containing media. Salvia miltiorrhiza was used to induce human BMSC (hBMSC) to differentiate. BMSC were identified with immunocytochemistry. Semi-quantitative RT-PCR was used to examine mRNA expression of neurofilamentl (NF1), nestin and neuron-specific enolase (NSE) in rat BMSC (rBMSC). Rat BMSC labelled by Hoschst33258 were transplanted into striatum of rats to trace migration and distribution.

Results: rBMSC expressed NSE, NF1 and nestin mRNA, and NF1 mRNA and expression was increased with induction of Salvia miltiorrhiza. A small number of hBMSC were stained by anti-nestin, anti-GFAP and anti-S100. Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. Some differentiated neuron-like cells, that expressed NSE, beta-tubulin and NF-200, showed typical neuron morphology, but some neuron-like cells also expressed alpha smooth muscle protein, making their neuron identification complicated. rBMSC could migrate and adapted in the host brains after being transplanted.

Conclusion: Bone marrow stromal cells could express phenotypes of neurons, and Salvia miltiorrhiza could induce hBMSC to differentiate into neuron-like cells. If BMSC could be converted into neurons instead of mesenchymal derivatives, they would be an abundant and accessible cellular source to treat a variety of neurological diseases.
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October 2006

[Biological characteristics of human umbilical cord-derived mesenchymal stem cells and their differentiation into neurocyte-like cells].

Zhonghua Er Ke Za Zhi 2006 Jul;44(7):513-7

Department of Pediatrics, 2nd Affiliated Hospital of Shantou University Medical College, Shantou 515041, China.

Objective: To investigate the isolation and expansion of mesenchymal stem cells (MSCs) from human umbilical cord Wharton's jelly and their biological identities, and explore the possibility of inducing human umbilical cord-derived MSCs to differentiate into neurocyte-like cells.

Methods: The growth and proliferative abilities of human umbilical cord-derived MSCs were observed, and their immunophenotypes were determined by flow cytometry. Salvia miltiorrhiza and beta-sulfhydryl alcohol were adopted to induce the cells to differentiate. The differentiated and undifferentiated cells were identified with immunocytochemistry. The pleiotrophin and nestin genes were measured by RT-PCR.

Results: A population of human umbilical cord-derived MSCs were isolated from human umbilical Wharton's jelly; they were processed to obtain a fibroblast-like population of cells and could be maintained in vitro for extended periods with stable population doubling, and they were expanded as undifferentiated cells in culture for more than 10 passages, indicating their proliferative capacity. The human umbilical cord-derived MSCs were positive for CD(29), CD(44), CD(59), CD(105), but negative or weakly expressed the markers of hematopoietic cells such as CD(14), CD(33), CD(34), CD(28), CD(45) and CD(117). The important GVHD correlation markers were negative or weakly expressed, including CD(80) (B7-1), CD(86) (B7-2), CD(40) and CD(40L). Salvia miltiorrhiza beta-sulfhydryl alcohol could induce the MSCs to express nestin, a marker of neuronal precursor stem cells at early stage of differentiation. Later, they exhibited neural phenotypes, expressing beta-tubulin III and neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that the MSCs could express pleiotrophin either before or after the induction of salvia miltiorrhiza, furthermore, after the induction the expression was markedly enhanced and the nestin gene was also expressed.

Conclusion: The human MSCs could be isolated from human umbilical cord Wharton's jelly, and it was easy to propagate these MSCs. The negative GVHD correlated markers might result from the fact that MSCs had no HLA barrier, which may suggest potential clinical significance. The MSCs are capable of differentiating into neurocyte-like cells and they may represent an alternative stem cell source for CNS cells transplantation.
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July 2006

Expression of hepatitis B virus genes in early embryonic cells originated from hamster ova and human spermatozoa transfected with the complete viral genome.

Asian J Androl 2006 May;8(3):273-9

Research Center for Reproductive Medicine, Cell Biology and Medical Genetics Department, Shantou University Medical College, 22 Xinling Road, Shantou 515041, China.

Aim: To detect the expression of hepatitis B virus (HBV) genes (HB S and C genes) in early embryonic cells after introducing motile human sperm carrying HBV DNA into zona-free hamster oocytes via the in vitro fertilization (IVF) technique.

Methods: Human sperm-mediated HBV genes were delivered into zona-free hamster oocytes by the IVF method. Polymerase chain reaction (PCR) was used to detect HB S and pre-Core/Core (pre-C/C) coding genes both in one- and two-cell embryos. Reverse transcription-PCR (RT-PCR) analysis was used to study the expression of the two genes. Fluorescence in situ hybridization (FISH) analysis using the full-length HBV DNA as the hybridization probe was performed to confirm the integration of viral DNA in the host embryonic genome.

Results: Both HB S and pre-C/C coding genes are present and transcribed in one- and two-cell embryos originated from hamster ova IVF with human spermatozoa carrying HBV DNA sequences.

Conclusion: Sperm-mediated HBV genes are able to replicate and express themselves in early embryonic cells. These results provide direct evidence that HBV DNA could transmit vertically to the next generation via the male germ line.
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http://dx.doi.org/10.1111/j.1745-7262.2006.00141.xDOI Listing
May 2006

Human umbilical cord Wharton's Jelly-derived mesenchymal stem cells differentiation into nerve-like cells.

Chin Med J (Engl) 2005 Dec;118(23):1987-93

Department of Pediatrics, Second Affiliated Hospital of Shantou University Medical College, Shantou 515041, China.

Background: The two most basic properties of mesenchymal stem cells (MSCs) are the capacities to self-renew indefinitely and differentiate into multiple cells and tissue types. The cells from human umbilical cord Wharton's Jelly have properties of MSCs and represent a rich source of primitive cells. This study was conducted to explore the possibility of inducing human umbilical cord Wharton's Jelly-derived MSCs to differentiate into nerve-like cells.

Methods: MSCs were cultured from the Wharton's Jelly taken from human umbilical cord of babies delivered after full-term normal labor. Salvia miltiorrhiza and beta-mercaptoethanol were used to induce the human umbilical cord-derived MSCs to differentiate. The expression of neural protein markers was shown by immunocytochemistry. The induction process was monitored by phase contrast microscopy, electron microscopy (EM), and laser scanning confocal microscopy (LSCM). The pleiotrophin and nestin genes were measured by reverse transcription-polymerase chain reaction (RT-PCR).

Results: MSCs in the Wharton's Jelly were easily attainable and could be maintained and expanded in culture. They were positive for markers of MSCs, but negative for markers of hematopoietic cells and graft-versus-host disease (GVHD)-related cells. Treatment with Salvia miltiorrhiza caused Wharton's Jelly cells to undergo profound morphological changes. The induced MSCs developed rounded cell bodies with multiple neurite-like extensions. Eventually they developed processes that formed networks reminiscent of primary cultures of neurons. Salvia miltiorrhiza and beta-mercaptoethanol also induced MSCs to express nestin, beta-tubulinIII, neurofilament (NF) and glial fibrillary acidic protein (GFAP). It was confirmed by RT-PCR that MSCs could express pleiotrophin both before and after induction by Salvia miltiorrhiza. The expression was markedly enhanced after induction and the nestin gene was also expressed.

Conclusions: MSCs could be isolated from human umbilical cord Wharton's Jelly. They were capable of differentiating into nerve-like cells using Salvia miltiorrhiza or beta-mercaptoethanol. The induced MSCs not only underwent morphologic changes, but also expressed the neuron-related genes and neuronal cell markers. They may represent an alternative source of stem cells for central nervous system cell transplantation.
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December 2005

Presence and integration of HBV DNA in mouse oocytes.

World J Gastroenterol 2005 May;11(19):2869-73

Research Center of Reproductive Medicine, Shantou University Medical College, 22 Xinling Road, Shantou 515041, Guangdong Province, China.

Aim: Hepatitis B is a worldwide public health problem. To explore the feasibility of hepatitis B virus (HBV) vertical transmission via oocytes, the presence and integration of HBV DNA in mouse oocytes were studied.

Methods: Genomic DNA was isolated and metaphases were prepared, respectively from mouse oocytes cocultured with pBR322-HBV DNA plasmids. PCR, Southern blot, dot hybridization and fluorescence in situ hybridization (FISH) were performed to explore the existence and integration of HBV DNA in oocytes.

Results: PCR detected positive bands in the tested samples, and then Southern blot revealed clear hybridization signals in PCR products. Final washing solutions were collected for dot hybridization and no signal for HBV DNA was observed, which excluded the possibility that contamination of washing solutions gave rise to positive results of PCR and Southern blot. FISH demonstrated that 36 of 1,000 metaphases presented positive signals.

Conclusion: HBV DNA sequences are able to pass through the zona and oolemma to enter into oocytes and to integrate into their chromosomes. HBV DNA sequences might be brought into embryo via oocytes as vectors when they are fertilized with normal spermatozoa.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4305652PMC
http://dx.doi.org/10.3748/wjg.v11.i19.2869DOI Listing
May 2005
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