Comparative Analysis of Protein Tyrosine Phosphatases Regulating Microglial Activation

Protein tyrosine phosphatases (PTPs) are key regulatory factors in inflammatory signaling pathways. Although PTPs have been extensively studied, little is known about their role in neuroinflammation. In the present study, we examined the expression of 6 different PTPs (PTP1B, TC-PTP, SHP2, MEG2, LYP, and RPTPβ) and their role in glial activation and neuroinflammation. All PTPs were expressed in brain and glia. The expression of PTP1B, SHP2, and LYP was enhanced in the inflamed brain. The expression of PTP1B, TC-PTP, and LYP was increased after treating microglia cells with lipopolysaccharide (LPS). To examine the role of PTPs in microglial activation and neuroinflammation, we used specific pharmacological inhibitors of PTPs. Inhibition of PTP1B, TC-PTP, SHP2, LYP, and RPTPβ suppressed nitric oxide production in LPS-treated microglial cells in a dose-dependent manner. Furthermore, intracerebroventricular injection of PTP1B, TC-PTP, SHP2, and RPTPβ inhibitors downregulated microglial activation in an LPS-induced neuroinflammation model. Our results indicate that multiple PTPs are involved in regulating microglial activation and neuroinflammation, with different expression patterns and specific functions. Thus, PTP inhibitors can be exploited for therapeutic modulation of microglial activation in neuroinflammatory diseases.

Effect of Cryopreservation on the Heat Shock Protein 90 Expression in Mouse Ovarian Tissue / 대한불임학회지

OBJECTIVE: Heat shock protein family is related to protective mechanism of cells by environmental changes. This study was performed to evaluate the effect of cryopreservation on the heat shock protein 90 (Hsp90) expression in mouse ovarian tissue. METHODS: Cryopreservation of mouse ovarian tissue was carried out by slow freezing method. The mRNA level of Hsp90 expression in both fresh and cryopreserved mouse ovarian tissue was analyzed by RT-PCR. The protein expression of Hsp90 was evaluated by Western blot analysis and immunohistochemistry. RESULTS: The mRNA and protein of Hsp90 were expressed in both fresh and cryopreserved mouse ovarian tissue. The amount of Hsp90 mRNA was increased in cryopreserved ovarian tissue after 60 and 90 minutes after thawing and incubation. The amount of Hsp90 protein was increased in the cryopreserved ovarian tissue after 6 hours of the incubation in Western blot analysis. In immunohistochemical study, Hsp90 protein was localized in cytoplasm of oocytes and granulosa cells. Significant level of immunoreactive Hsp90 protein was detected in theca cells contrast to the weak expression in ovarian epithelial cells. CONCLUSION: This results showed the increase of Hsp90 expression in both mRNA and protein level in the cryopreserved mouse ovarian tissue. It can be suggested that Hsp90 may play a role in the protective or recovery mechanism against the cell damage during cryopreservaion.

Clinical application of preimplantation genetic diagnosis (PGD) using fluorescence in-situ hybridization to balanced reciprocal or Robertsonian translocation carriers in human IVF-ET program / 대한산부인과학회잡지

OBJECTIVE: This study was performed to evaluate the efficiency of preimplantation genetic diagnosis (PGD) using fluorescence in-situ hybridization (FISH) in Robertsonian or balanced reciprocal translocation carriers in human IVF-ET programm. METHOD: FISH was carried out in 25 cycles of 15 couples. Two-color FISH analysis was performed on 54 polar bodies in 3 cycles and 234 blastomeres in 22 cycles. After FISH analysis, the embryos with normal FISH signals were transferred into mother's uterus. RESULTS: In FISH analysis of polar bodies, 18 nuclei of polar bodies were normal and 12 embryos were transferred in 3 cycles. FISH efficiency per oocyte was 95.0% in cases using polar bodies. In FISH analysis of blastomeres, 49 embryos were normal and transferred in 21 cycles. FISH efficiency per embryo was 92.7% using blastomeres. At present, three pregnancies were achieved. A girl and a boy were delivered. Both of them were translocation carriers. The other conceptus showed normal karyotype. CONCLUSIONS: According to this study, PGD using FISH can be successfully applied for the patients with translocations of chromosomes.

Analysis of Inactivating Point Mutation of the Follicle Stimulating Hormone Receptor Gene in Korean Infertile Men / 대한남성과학회지

PURPOSE: Follicle stimulating hormone (FSH) is essential for normal gametogenesis. In females FSH is required for ovarian development and follicle maturation whereas in males FSH determines Sertoli cell number and normal spermatogenesis quantitatively and qualitatively. Recently, Tapanainen et al. (1) reported that an anactivating point mutation (C566T) of the FSH receptor gene in males suppressed spermatogenesis but did not cause azoospermia or absolute infertility. To study the significance of the C566T inactivating point mutation in male infertility, we examine the FSH receptor gene in men with azoospermia or oligozoospermia. MATERIALS AND METHODS: Peripheral blood was collected from each patient who had elevated serum FSH. To amplify a suitable segment of the FSHR gene containing nuceotide 566, primer flanking the region was used. And to screen individuals for the C566T mutation, PCR was performed for exon 7 of the FSH receptor gene in 58 patients. RESULTS: The 78-bp fragment containing nucleotide 566 was present in all patient, the PCR product in cleaved into fragments 51-bp and 27-bp by Bsm I digestion. No inactivating point mutations of FSH receptor gene was identified in men with azoospermia or oligozoospermia. CONCLUSIONS: Inactivating point mutation (C566T) of the FSH receptor is not a common cause of male infertility. However we cannot exclude point mutations in other regions of the FSH receptor gene in some patient with azoospermia or oligozoospermia.

A study on the patterns of expression of the DAZ and HSP genes in the testicular tissue of men with azospermia

In order to examine whether microdeletions on the Y chromosome exist or not and observe the aspects of expression of DAZ which is suggested to be essential in spermatogenesis in testicular tissue, tissues of 21 patients with azospermia were analyzed by using PCR methods and reverse transcription-PCR. The primers used for the analysis of the microdeletions on the Y chromosome were gene-specific. According to the results of the PCR with genomic DNA of the peripheral blood extracted from each patient, of the 21 men with azospermia 2 displayed microdeletions of the DAZ gene in the Y chromosome but none of HSP70A and HSP70B. And the reverse transcription-PCR of the RNA extracted from the testicular tissue of the patients gave results which found no amplified products of the mRNA of DAZ in the patients with microdeletions of that gene as expected, and confirmed patterns of expression of the mRNA of HSP70B in 5 whereas none could be seen with regard to HSP70A. Furthermore, the sperm of these 5 men were discovered t In accordance with the results previously described, one can see that the microdeletions of DAZ are associated with spermatogenesis and contemplate that HSP70B plays an important part in the maturation process of sperm. But it is considered that there is no correlation between the genes DAZ and HSP and since factors associated with the process of spermatogenesis are being continously discovered more studies on this should be advanced.

Analysis of the Azoospermia Factor (AZF) Gene on Y Chromosome and Expression Pattern of DAZ Gene in Korean Infertile Men / 대한불임학회지

Cytogenetic observations of loss of the distal portion of the Y chromosome long arm were found to be associated with disrupted spermatogenesis. The existence of a gene involved in the regulation of spermatogenesis, the azoospermia factor (AZF), was postulated. In this study, we screened the AZF region including DAZ and DAZH genes and observed the expression pattern of DAZ and DAZH transcript in infertile men with azoospermia and oligospermia by using a sequence-tagged site (STS)-based PCR method. PCR primers were synthesized for 11 STSs that span Yq interval 6, SRY, DAZ, and DAZH, functional DAZ homologue on chromosome 3. Microdeletions were detected in 4/32 (12.5%) azoospermic men and 1/11 (9%) severe oligospermic men. Only 2 of 5 patients had microdeletions of Yq that contained the 342 gene, whereas the other 3 patients had deletions extending from intervals 5L-6F proximal to the DAZ gene on Yq. Testis biopsies of the azoospermic patients revealed a variety from Sertoli cell-only syndrome to testicular maturation arrest. Of 4 men with clinical data available, average testis size was R: 13.8 co, L: 13.8 co, serum T was 4.0+/-1.25 ng/ml, LH was 3.63+/-1.90 mIU/ml, and FSH was 8.85 +/- 5.13 mIU/ml. These values did not differ significantly from the remainder of the patients tested. We could not observed the DAZ transcript in 2 patients, who have no mature spermatozoa. In 11,6% of patients microdeletions of the AZF could be detected. These deletions in the AZF region seem to be involved causing spermatogenic failure. But the frequency of microdeletions proximal to DAZ suggests that DAZ is not the only gene associated with spermatogenic failure.