Dendritic cell TLR4 induces Th1-type immune response against Cryptosporidium parvum infection.

The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 µg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

Dendritic cell TLR4 induces Th1-type immune response against Cryptosporidium parvum infection

@#The objective of this study was to investigate the mechanism of Toll-like receptor (TLR4)- mediated dendritic cell (DC) immune against Cryptosporidium parvum infection. C. parvum sporozoites were labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester. Murine bone marrow-derived DCs were isolated, and divided into TLR4 antibody blocking (TAB; infected with 2 × 105 labeled sporozoites and 0.5 μg TLR4 blocking antibody), TLR4 antibody unblocking (TAU; infected with 2 × 105 labeled sporozoites), and blank control (BC; with 1.5 mL Roswell Park Memorial Institute 1640 medium) groups. The adhesion of Cryptosporidium sporozoites to DCs and CD11c+ levels were examined by fluorescence microscopy and flow cytometry. Male KM mice were orally injected with C. parvum. The proliferation of T lymphocytes in spleen, expression of cytokines in peripheral blood, and TLR4 distribution features in different organs were further determined by immunohistochemistry. A significantly higher expression of CD11c+ and higher C. parvum sporozoite adhesion were found in the TAU group compared with other groups. The expression of CD4+CD8- /CD8+CD4- in the spleen were obviously differences between the TAB and TAU groups. The expression of TLR4, interleukin IL-4, IL-12, IL-18 and IFN-γ improved in the TAU group compared with TAB group. Higher expression of TLR4 was detected in the lymph nodes of mice in the TAU group, with pathological changes in the small intestine. Hence, TLR4 could mediate DCs to recognize C. parvum, inducing Th1 immune reaction to control C. parvum infection.

Missense mutation in DNAJB13 gene correlated with male fertility in asthenozoospermia.

BACKGROUND: The most common type of male infertility is asthenospermia. We cloned DnaJ heat shock protein family member B13 (Dnajb13/DNAJB13), a type II HSP40 family member that is highly expressed in the testis. DNAJB13 plays a crucial role in sperm flagellar function. OBJECTIVES: The aim of this study was to investigate whether a correlation exists between DNAJB13 and low sperm motility in infertile men. MATERIALS AND METHODS: In the present study, we performed a mutation screening of the DNAJB13 gene in 92 idiopathic asthenozoospermia patients and 200 men with normal fertility. Additionally, we used immunoelectron microscopy, co-immunoprecipitation, mass spectrometric detection, indirect immunofluorescence assay, transmission electron microscopy studies, isobaric tags for relative and absolute quantitation, and multiple reaction monitoring studies to analyze changes in DNAJB13 protein. RESULTS: A novel c.106T>C mutation of DNAJB13 was present in nearly 10% (9/92) of idiopathic asthenozoospermia patients and was absent in 200 fertile men. A computer-assisted sperm analyzer and transmission electron microscopy analysis using samples from 9 patients with DNAJB13 mutations demonstrated that most spermatozoa were immotile due to sperm tail defects. Multiple reaction monitoring results indicated that DNAJB13 protein levels were reduced after gene mutation. We achieved a pregnancy rate of 100% in 8 patients with DNAJB13 mutations using ICSI. DISCUSSION AND CONCLUSION: The DNAJB13 heterozygous variant may affect fertility. ICSI can help these patients with low fertility to father children.

Whole-exome sequencing identifies a homozygous donor splice-site mutation in STAG3 that causes primary ovarian insufficiency.

Primary ovarian insufficiency (POI) is the depletion or loss of normal ovarian function, which cause infertility in women before the age of 40 years. Two homozygous germline truncation mutations in STAG3 gene had been reported to causes POI in consanguineous families. Here, we aimed to identify the genetic cause of POI in 2 affected sisters manifested with primary amenorrhea and partial development of secondary sexual characters with normal range of height of a consanguineous Han Chinese family. Whole-exome and Sanger sequencing identified a homozygous donor splice-site mutation (NM_012447.2: c.1573+5G>A) in the STAG3 gene. RT-PCR revealed that the mutation causes loss of wild-type donor splice-site which leads to aberrant splicing of STAG3 mRNA and consecutive formation of STAG3 alternative transcript (p.Leu490Thrfs*10) . This is the first report of splice-site mutation of STAG3 gene causes POI in 2 Han Chinese patients.

Time-lapse observation and transcriptome analysis of a case with repeated multiple pronuclei after IVF/ICSI.

PURPOSE: The purpose of this study was to investigate the cause of repeated multipronucleus (MPN) formation in zygotes in a patient after both in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). METHOD: This is a case study. A patient had unexplained primary infertility with recurring total MPN zygotes after IVF and ICSI cycles. Time-lapse monitoring of pronucleus formation was carried out. Embryos developed from MPN zygotes were analyzed by fluorescence in situ hybridization (FISH). Single-cell RNA-seq analysis was used to identify gene expression profiles of the patient's oocyte and zygote, and these were compared to the data from oocytes and zygotes from donors with normal fertilization (patient, n = 1; donors, n = 4). Oocyte-specific genes with differential expression were selected by the Amazonia! RESULTS: From time-lapse analysis, we observed the formation of multiple micronuclei near the site of the second polar body extrusion. These micronuclei migrated, expanded, and juxtaposed with the male pronucleus leading to a multipronucleus. None of these MPN zygotes could develop to the blastocyst stage, and FISH analysis revealed a chaotic chromosomal complement in the arrested embryos. RNA-seq analysis showed 113 differentially expressed genes (DEGs) between the patient and the donor oocytes and zygotes. Moreover, 25 of the 113 DEGs were unique or highly expressed in oocytes and early embryos. From 25 DEGs, three genes, DYNC2LI1, NEK2, and CCNH, which are involved in meiosis and the chromosome separation process, were further validated by real-time PCR. CONCLUSION: We identified several candidate genes affecting pronucleus formation as a new cause of infertility.

Cleavage pattern predicts developmental potential of day 3 human embryos produced by IVF.

Time-lapse technique provides opportunities to observe the dynamic process of human early development. Previous studies have suggested several abnormal division patterns were associated with decreased developmental potential, but no systematic results are currently available. In this study, seven abnormal division patterns were observed during early cleavage, and these had different effects on the further development potential of daughter blastomeres. According to the severity and occurrence of abnormal division patterns during the initial three cleavages, an embryo hierarchical classification model was developed and day 3 embryos were classified into six grades (from A to F). The good-quality blastocyst formation rate for these grades decreased from 70.8-3.8% (P < 0.001). In a prospective observational study, 139 IVF cycles were recruited to assess the efficiency of this classification model. In the embryos that had confirmed implantation results, the implantation rate decreased from 67.0% (Grade A) to 0% (Grade D;P < 0.001). These results indicated that cleavage patterns can predict the developmental potential of day 3 human embryos.

Using SNP array to identify aneuploidy and segmental imbalance in translocation carriers.

Translocation is one of the more common structural rearrangements of chromosomes, with a prevalence of 0.2%. The two most common types of chromosomal translocations, Robertsonian and reciprocal, usually result in no obvious phenotypic abnormalities when balanced. However, these are still associated with reproductive risks, such as infertility, spontaneous abortion and the delivery of babies with mental retardation or developmental delay. In recent years, array-based whole-genome amplification (WGA) technologies, including microarray comparative genomic hybridization (array CGH; aCGH) and single-nucleotide polymorphism (SNP) micro-arrays, have enabled the screening of every chromosome for whole-chromosome aneuploidy and segmental imbalance. These techniques have been shown to have clinical application for translocation carriers. Promising studies have indicated that array-based PGD of translocation carriers can lead to transfer pregnancy rates of 45-70% [2]. In addition to genetic testing techniques, the embryo biopsy stage (polar body, cleavage embryo or blastocyst) and the mode of embryo transfer (fresh or frozen embryos) can affect the outcome of PGD. It is now generally recommended that blastomere biopsy should be replaced by blastocyst biopsy to avoid a high mosaic rate and biopsy-related damage to cleavage-stage embryos, which might affect embryo development. However, more clinical data are required to confirm that the technique of SNP array-based PGD (SNP-PGD) combined with trophectoderm (TE) biopsy and frozen embryo transfer (FET) is superior to traditional FISH-PGD combined with Day 3 (D3) blastomere biopsy and fresh embryo transfer.

Taxonomy and epidemiology of Mucor irregularis, agent of chronic cutaneous mucormycosis.

Mucormycosis usually presents as a progressive infection with significant angio-invasion. Mucormycosis due to Mucor irregularis (formerly Rhizomucor variabilis var. variabilis), however, is exceptional in causing chronic cutaneous infection in immunocompetent humans, ultimately leading to severe morbidity if left untreated. More than 90 % of the cases known to date were reported from Asia, mainly from China. The nearest neighbour of M. irregularis is the saprobic species M. hiemalis. The aim of this study was to evaluate the taxonomic position, epidemiology, and intra- and inter-species diversity of M. irregularis based on 21 strains (clinical n = 17) by multilocus analysis using ITS, LSU, RPB1 and RPB2 genes, compared to results of cluster analysis with amplified fragment length polymorphism (AFLP) data. By combining MLST and AFLP analyses, M. irregularis was found to be monophyletic with high bootstrap support, and consisted of five subgroups, which were not concordant in all partitions. It was thus confirmed that M. irregularis is a single species at 96.1-100 % ITS similarity and low recombination rates between populations. Some geographic structuring was noted with some localised populations, which may be explained by limited air-dispersal. The natural habitat of the species is likely to be in soil and decomposing plant material.

Single-nucleotide polymorphism microarray-based preimplantation genetic diagnosis is likely to improve the clinical outcome for translocation carriers.

STUDY QUESTION: Is preimplantation genetic diagnosis (PGD) for translocation carriers more effective when done with a single-nucleotide polymorphism (SNP) array using trophectoderm (TE) biopsy and frozen embryo transfer (FET) compared with traditional PGD based on fluorescence in situ hybridization (FISH-PGD) using blastomere biopsy and fresh embryo transfer? SUMMARY ANSWER: The procedure using the SNP array combined with TE biopsy and FET significantly improves the clinical pregnancy rate for translocation carriers. The miscarriage rate also slightly decreases. WHAT IS KNOWN ALREADY: FISH-PGD has been widely used in translocation carriers but the clinical outcomes have not been ideal. SNP arrays can detect both chromosome segmental imbalances and aneuploidy, and may overcome the limitations of FISH in PGD for translocation carriers. STUDY DESIGN, SIZE AND DURATION: This was a retrospective study of 575 couples with chromosomal translocations, including 169 couples treated by SNP-PGD between October 2011 and August 2012, and 406 couples treated by FISH-PGD between January 2005 and October 2011. PARTICIPANTS/MATERIALS, SETTING, METHODS: The study was set in an IVF center at the Reproductive and Genetic Hospital of CITIC-Xiangya, China. In total, 169 couples underwent SNP analysis, including 52 Robertsonian translocation carriers and 117 carriers of reciprocal translocations. Blastocysts (n = 773) were biopsied and FET was carried out on the balanced embryos. Four hundred and six couples underwent FISH-PGD, including 149 Robertsonian translocation carriers and 257 reciprocal translocation carriers. In total, 3968 embryos were biopsied and balanced embryos were transferred fresh. The SNP-PGD results and clinical outcomes were compared with those of FISH-PGD. MAIN RESULTS AND THE ROLE OF CHANCE: Reliable SNP-PGD results were obtained for 717 out of 773 (92.8%) biopsied blastocysts. The proportions of normal/balanced embryos, embryos with translocation-related and translocation-unrelated abnormalities, the median number of embryos per patient, the ongoing pregnancy rate per embryo transfer and the miscarriage rate were 58, 23, 19, 2, 69 and 12%, respectively, for Robertsonian translocation carriers and 36, 52, 12, 1, 74 and 11%, respectively, in reciprocal translocation carriers. Reliable FISH-PGD results were obtained for 3452 out of 3968 (87.0%) biopsied embryos. The proportions of normal/balanced embryos, unbalanced embryos, the median number of embryos per patient, the ongoing pregnancy rate per transfer and the miscarriage were 36, 64, 3, 38 and 17%, respectively, for Robertsonian translocation carriers and 20, 80, 1, 39 and 16%, respectively, for reciprocal translocation carriers. Thus, SNP-PGD achieved a higher pregnancy rate but a lower miscarriage rate than FISH-PGD. There were no significant differences in maternal age, basal endocrine level and the average number of retrieved oocytes and good-quality D3 embryos in the SNP-PGD group compared with the FISH-PGD group. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study with the two groups treated in different periods; therefore, there is a chance of sample bias and a possibility that the results were influenced by other factors that changed over time. Furthermore, the two treatment protocols differ in several respects and we cannot say which makes the greatest contribution to the difference in success. Complete pregnancy outcomes of SNP-PGD have not been obtained as some embryos have not been transferred yet. We cannot exclude differences between the final data and the data in the present manuscript. WIDER IMPLICATIONS OF THE FINDINGS: The adoption of SNP-PGD combined with TE biopsy and FET may significantly improve the clinical pregnancy rate, and decrease the miscarriage rate after PGD for translocation carriers.

Value of transvaginal sonography in diagnosing heterotopic pregnancy after in-vitro fertilization with embryo transfer.

OBJECTIVES: To assess the value of transvaginal sonography (TVS) in the diagnosis of heterotopic pregnancy (HP) in the first trimester after in-vitro fertilization with embryo transfer (IVF-ET). METHODS: This was a retrospective review of women undergoing IVF-ET between January 2005 and December 2011. Women were diagnosed with an HP using TVS if a visible intrauterine gestational sac was observed with any of the following: (i) an inhomogeneous adnexal mass; (ii) an empty extrauterine gestational sac seen as a hyperechoic ring; or (iii) a yolk sac and/or fetal pole with or without cardiac activity in an extrauterine sac. RESULTS: Overall, 16 483 consecutive women who underwent IVF had TVS during the study. Of these, 174 cases were diagnosed on TVS as having an HP, and 10 cases were missed. Fifty-two cases were treated expectantly and were excluded from the analysis. Three types of ultrasonographic presentation of ectopic pregnancy (EP) were seen in HP patients, with a gestational sac found in 70 cases, a ring sign in 21 and an adnexal mass in 31. The sensitivity and specificity of TVS for the detection of HP were 92.4 and 100%, respectively, with positive and negative predictive values of 100 and 99.9%. The HP cases comprised 103 tubal EPs and 29 non-tubal EPs. In 93 patients (70.5%), their intrauterine pregnancy resulted in a live birth, 37 patients (28.0%) suffered an early miscarriage and two patients (1.5%) had a late miscarriage. CONCLUSION: Early TVS performed by an experienced sonographer has a high sensitivity for making the correct diagnosis of HP after IVF-ET.

Cyclic regulation of LPA3 in human endometrium.

BACKGROUND: Lysophosphatidic acid (LPA) belongs to the group of lipid messengers, which plays a pivotal role in the establishment of implantation via its cellular receptor, LPA3. The aims of the present study were to characterize LPA3 mRNA and protein in human endometrium during the normal human menstrual cycle. METHODS: Forty-three normally cycling volunteers without reproductive disorders were randomized to undergo endometrial sampling on a specific cycle day. Samples were assessed for relative LPA3 mRNA expression using real-time PCR and for LPA3 protein using immunohistochemistry and western blot. RESULTS: The expression of LPA3 mRNA significantly increased during the early and late secretory phase compared with other menstrual phases. LPA3 protein was localized to the epithelial and stromal cells and expression levels followed the same pattern as for LPA3 mRNA. CONCLUSION: In the normal human menstrual cycle, LPA3 mRNA and protein expression also change, indicating that this gene may be related to the function of the endometrium.

Tumourigenic characteristics of embryonal carcinoma cells as a model for studying tumour progression of human embryonic stem cells.

OBJECTIVES: The objective of this study was to compare tumourigenic characteristics of human embryonic stem cells (HESCs) and embryonal carcinoma cells (ECCs) to identify a robust and simple model for studying certain aspects of cell transformation and tumourigenesis, in tumour progression of HESCs. MATERIALS AND METHODS: SSEA-3 positive ECCs (NTERA-2) cells were identified and compared to HESCs (ch HES-20) in terms of pluripotency and differentiation capacity, growth characteristics, gene expression profiles and signalling pathways. RESULTS: Our results showed that NTERA-2 cells shared similarities in expression markers of pluripotency to ch HES-20 cells. However, NTERA-2 cells also expressed some markers of differentiation and had a tendency to differentiate towards ectodermal endpoints. We identified NTERA-2 cells with higher S-phase fraction in cell cycle distribution, anti-apoptosis markers and robust self-renewal ability, compared to ch HES-20 cells. Microarray analysis and real-time PCR results showed that some oncogenes were up-regulated and tumour-suppression genes were down-regulated, whereas pluripotency-related genes were up-regulated and differentiation-related genes were down-regulated, and that Wnt and Notch signalling pathways were activated during progression from ES cells to EC cells. CONCLUSION: Tumourigenic characteristics of ECCs may provide a valuable insight into possible tumour progression of HESCs.

Differences between karyotypically normal and abnormal human embryonic stem cells.

OBJECTIVES: To compare different biological characteristics of human embryonic stem cells (HESCs) between those with normal and those with abnormal karyotype. MATERIALS AND METHODS: Culture-adapted HESCs (chHES-3) with abnormal karyotype were compared with karyotypically normal cells, with regard to pluripotency and differentiation capacity, ultrastructure, growth characteristics, gene expression profiles and signalling pathways. RESULTS: We found a new abnormal karyotype of HESCs. We observed that chHES-3 cells with normal and abnormal karyotypes shared similarities in expression markers of pluripotency; however, karyotypically abnormal chHES-3 cells had a tendency for differentiation towards ectoderm lineages and were easily maintained in suboptimal culturing conditions. Abnormal chHES-3 cells displayed relatively mature cell organelles compared to normal cells, and karyotypically abnormal chHES-3 cells had increased survival and population growth. Genes related to cell proliferation and apoptosis were up-regulated, but genes associated with genetic instability (p53, Rb, BRCA1) were down-regulated in the karyotypically abnormal cells. CONCLUSION: Karyotypically abnormal chHES-3 cells had a more developed capacity for proliferation, resistance to apoptosis and less genetic stability compared to normal chHES-3 cells and may be an excellent model for studying and characterizing initial stages that determine transition of embryonic stem cells into cancer stem cells.

Association of AR rs6152G/A gene polymorphism with susceptibility to polycystic ovary syndrome in Chinese women.

Hyperandrogenism is a central feature of polycystic ovary syndrome (PCOS). Androgens act via the androgen receptor (AR). The rs6152G/A AR gene polymorphism has been reported to be associated with male pattern baldness (MPB), which is a common characteristic of males in PCOS families. Therefore, we investigated the relationship between the rs6152G/A polymorphism and PCOS in Han Chinese women. The rs6152G/A AR gene polymorphism was screened by restriction fragment length polymorphism (RFLP) in 224 PCOS women and 223 control subjects from the Reproductive and Genetic Hospital of CITIC-XIANGYA in China. There was a significantly higher prevalence of genotypes containing the A allele in PCOS patients compared with controls (P < 0.05). Patients carrying the rs6152A allele had a 1.608-fold greater risk of developing polycystic ovary syndrome compared with rs6152GG homozygotes (OR = 1.608, CI = 1.008-2.597, P < 0.05). In conclusion, the individuals carrying the rs6152A allele had significantly higher susceptibility to polycystic ovary syndrome than those that were GG homozygotes.

Cloning, characterization and identification of Rcet1-v1 and Rcet1-v2, two novel splice variants of mouse Rcet1 related to Cres subgroup of family 2 cystatins.

Cystatins are physiological cysteine proteinase inhibitors. We used digital differential display (DDD) to clone two novel splice variants Rcet1-v1 and Rcet1-v2 which were isolated from adult mouse testis cDNA library. Sequence analysis revealed that Rcet1-v1 and Rcet1-v2 cDNAs are 454 and 610 bp in length, respectively, and each has four exons, but the lengths of their second and third exons are different, with the results that these cDNAs encoded two different putative proteins. The deduced proteins were 88 amino acid residues (RCET1-v1) and 140 residues (RCET1-v2) in length and have one potential signal peptide and one cystatin domain, respectively, but lack part critical consensus sites important for cysteine protease inhibition. These characteristics are seen in CRES subgroup, which related to the family 2 cystatins and primarily expressed in reproductive tract. RT-PCR analysis showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse testis, epididymis and cerebrum, but higher in testis than in epididymis and cerebrum. RT-PCR analysis also showed that Rcet1-v1 and Rcet1-v2 were specifically expressed in adult mouse pituitary and spermatogonium, but not expressed in spermatozoa. Results of in situ hybridization showed that Rcet1 gene expressed abundantly in mouse spermatogonium, spermatocytes and round spermatids; did not expressed in spermatozoa. At mouse testis different development stages, Rcet1-v1 and Rcet1-v2 were expressed very low from postnatal 1 day to postnatal 3 weeks; after postnatal 4 weeks, expressed steadily increased from postnatal 4 to 7 weeks, highest in postnatal 7 to 8 weeks, then keeping on the expressing level of postnatal 6 weeks in postnatal 13-57 weeks. All these indicated that Rcet1-v1 and Rcet1-v2 primarily expressed in mouse male reproductive tract and may play important roles in mouse spermatocytes and round spermatid development. Rcet1-v1 and Rcet1-v2 may be new members of Cres subgroup of the family 2 cystatins.

How medical ethical principles are applied in treatment with artificial insemination by donors (AID) in Hunan, China: effective practice at the Reproductive and Genetic Hospital of CITIC-Xiangya.

This paper investigates the efficiency of application of medical ethics principles in the practice of artificial insemination by donors (AID) in China, in a culture characterised by traditional ethical values and disapproval of AID. The paper presents the ethical approach to AID treatment as established by the Reproduction and Genetics Hospital of CITIC-Xiangya (CITIC Hunan-Yale Approach) in the central southern area of China against the social ethical background of China and describes its general features. The CITIC-Xiangya Approach facilitates the implementation of ethical relations between clinicians and patients participating in AID treatment procedures in Hunan-Yale.

Cloning of a novel gene, Cymg1, related to family 2 cystatins and expressed at specific stages of mouse testis development.

We have cloned a novel gene, Cymg1 (GenBank accession number AY600990), from a mouse testis cDNA library. Cymg1 is located in 2G3 of mouse chromosome 2. The cDNA includes an open reading frame that encodes 141 amino acid residues. The encoded polypeptide has a cysteine protease inhibitor domain found in the family 2 cystatins but lacks critical consensus sites important for cysteine protease inhibition. These characteristics are seen in the proteins of the CRES subfamily of the family 2 cystatins which are expressed specifically in the reproductive tract. CYMG1 protein shows 44% identity with mouse CRES and 30% identity with mouse cystatin C. Northern blot analysis showed that the Cymg1 gene was specifically expressed in adult mouse testis. RT-PCR also showed that Cymg1 was expressed in testis and spermatogonial cells. Cymg1 expression level varied in the different developmental stages of mouse testis, and were coincidental with spermatogenesis and sex maturation. These results indicate that Cymg1 may play important roles in mouse spermatogenesis and sex maturation.

[Rapid identification of human testis spermatocyte apoptosis-related gene, TSARG2, by nested PCR and draft human genome searching].

Cloning apoptosis-related novel genes is a key to further understanding of apoptosis mechanism and the biology process of germ cells, and is of momentous significance on clarifying physiological and pathological process of spermatogenesis. To rapidly attain human novel gene full-length cDNA sequence, the gene-specific primers and the vector-specific primers were designed for nested PCR, and draft human genome searching was performed to rapidly identify the TSARG2 (GenBank accession number AY040204) 5' end from a human testis cDNA library, by using a cDNA fragment (GenBank accession number BE644542) as an electronic probe, which was significantly changed in cryptorchidism and represented a novel gene. Furthermore, a mouse homologue of this gene was identified (GenBank accession number AF395083) by lab on-line. TSARG2 with a 1 233 bp length was composed of 6 exons and spanned about 115 kb of genomic DNA, The putative protein encoded by this gene was 305 amino acid with a theoretical molecular weight of 34 751 dalton and did not share significant homology with any known protein in databases. TSARG2 was expressed in many tissues and mapped to chromosome 4q33-34.1 by database analyses. Therefore, we propose that nested-PCR and draft human genome searching are rapid, sensitive, accurate and efficient method for isolating gene 5' end, even full-length gene from cDNA library.

[Localization and position effect of human APPSWE gene on transgenic mice by means of in situ fluorescence hybridization].

In situ fluorescence hybridization (FISH) was used to study the integration and localization of human APPSWE gene on chromosomes of transgenic mice. It was found that in 2 founder mice, 34 and 36 metaphases of 80 metaphases observed had shown obvious specific hybridizing signals, the detection rates were 42.5% and 45%, respectively; meanwhile, in 1 F1 and 1 F2 mice, 33 and 30 metaphases out of 100 metaphases observed had specific hybridizing signals, the detection rates were 33% and 30%, respectively. The transgenes were localized on chromosome 8, 1, 17 and 2. The results indicated that transgene APPSWE were integrated stably into the chromosomes of transgenic mice and could be transmitted to offsprings through germ cells. The transgenes were randomly integrated into multiple sites of mice chromosomes. Meanwhile, the phenotypes of transgenic mice were also studied and different integrated sites had obvious effects on the pheonotypes.

[Establishment of two-dimensional gel electrophoretic protein map of human spermatozoa].

OBJECTIVE: To establish the method of 2-dimensional electrophoresis(2-DE) for proteins of human spermatozoa and to construct a protein map of human spermatozoa. METHODS: The sperm pellet was prepared with simple Percoll layer protocol. We studied the effects of various sample preparation methods, loading quantities and isoelectric-focusing protocols on the quality of silver-stained 2-DE map, and constructed a primary protein map of human spermatozoa. RESULT: Up to 703 protein spots were acquired with sample preparation Method I while only 194-210 spots with Method II. With immobilized pH gradients and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(IPG-DALT) we could acquire over 700 spots while only 280-300 with isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(ISO-DALT). CONCLUSION: It is satisfactory to lyse sperm with sample preparation Method I and to separate sperm proteins by IPG-DALT for establishing 2-D map of human sperm.