Plac1 Expression Pattern at the Mouse Fetomaternal Interface and Involvement in Trophoblast Differentiation.

BACKGROUND/AIMS: It is well known that Plac1 is a placenta-specific gene; however, its spatiotemporal expression pattern and exact role at t h e mouse fetomaternal interface r e m a i n s unclear. METHODS: In situ hybridization (ISH) was used to localize the Plac1 mRNA at the mouse fetomaternal interface. A trophoblast stem cell (TS) differentiation model with Plac1 shRNA-overexpressing lentivirus was employed to investigate the possible role of Plac1 in placentation. Real-time RT-PCR was used to detect changes in gene expression. RESULTS: Plac1 was exclusively expressed in the ectoplacental cone (EPC) as well as in 8.5 and 9.5 days post-coitum (dpc) embryos. Subsequently, Plac1 expression was abundant in the spongiotrophoblast layer and moderately in the labyrinth layer until 13.5 dpc, and declined thereafter. Interestingly, Plac1 was also expressed by secondary trophoblast giant cells and glycogen trophoblast cells, but not in primary trophoblast giant cells. Plac1 transcription was increased during the TS differentiation (P < 0.01), and knockdown of Plac1 significantly impaired TS differentiation. CONCLUSION: Plac1 is abundantly expressed at the fetomaternal interface and in all trophoblast subtypes except in primary trophoblast giant cells. Plac1 knockdown retarded the progress of TS differentiation, indicating that Plac1 is necessary for normal trophoblast differentiation into various trophoblast subpopulations.

R383C mutation of human CDC20 results in idiopathic non-obstructive azoospermia.

Idiopathic azoospermia (IA) is a severe form of male infertility due to unknown causes. To investigate relative gene expression in human idiopathic non-obstructive azoospermia, we sequenced all the exons of cell division cycle 20 (CDC20) in 766 patients diagnosed with IA, as well as in 521 normally fertile men. Three novel missense mutations (S72G, R322Q, R383C) of CDC20 were detected and further confirmed by Sanger sequencing. The mRNA levels of securin, cyclin B, cyclin dependent kinase 1 (CDK1), and cyclin dependent kinase 2 (CDK2), which are all targeted for destruction via the anaphase-promoting complex/cyclosomeCDC20 (APC/CCDC20) pathway, were detected at relatively high levels using real-time quantitative polymerase chain reaction analysis. This demonstrated that the CDC20 R383C mutation led to dysfunction during the transition from metaphase to anaphase and facilitation of mitotic exit in vitro, and caused prolonged mitotic arrest during the cell cycle. This study suggests that a CDC20 R383C mutation may result in the pathogenesis of human IA.

PLAC1 is involved in human trophoblast syncytialization.

Placenta specific protein 1 (PLAC1) is thought to be important for murine and human placentation because of its abundant expression in placenta; however, the trophoblast subtypes that express PLAC1 at the fetomaternal interface and the major role of PLAC1 in placentation are still unclear. This study investigated the expression pattern of PLAC1 at the human fetomaternal interface and its involvement in trophoblast syncytialization. Localization of PLAC1 at the fetomaternal interface was studied using in situ hybridization (ISH) and immunohistochemistry (IHC) assays. Real time RT-PCR and Western Blot were employed to exhibit the expression pattern of PLAC1 during human spontaneous syncytialization of term primary cytotrophoblast cells (CTBs). Spontaneous syncytialization of a primary term CTBs model transfected with siRNA specific to PLAC1 was used to investigate the role of PLAC1 during human trophoblast syncytialization. The results showed that PLAC1 was mainly expressed in the human villous syncytiotrophoblast (STB) layer throughout gestation, and the expression level of PLAC1 was significantly elevated during human trophoblast syncytialization. Down-regulation of PLAC1 via specific PLAC1 siRNA transfection attenuated spontaneous syncytialization of primary term CTBs (p<0.05) as indicated by cell fusion index and the expression patterns of the corresponding markers. These data demonstrate the facilitative role of PLAC1 in normal human trophoblast syncytialization.

Comparison of different counting chambers using a computer-assisted semen analyzer.

A routine computer-assisted sperm analysis is an important diagnostic test in the andrology laboratory. To evaluate the accuracy and precision of the different types of counting chambers for human semen analysis in combination with a computer-assisted semen analyzer (CASA), a quality-control study that compared human sperm analysis results obtained using different counting chambers (Makler chamber, disposable 8-cell GoldCyto chamber, disposable 4-cell Leja chamber, a plain glass slide, and a tissue culture dish cover with a 24 × 24 mm(2) coverslip) in conjunction with the CASA systems was performed. Significantly higher counts of sperm concentration were obtained from the reusable Makler chamber than from the other counting chambers. Sperm motility from drop loaded counting chambers was significantly higher than that of capillary-loaded chambers. A plain glass slide and a tissue culture dish cover used with a coverslip showed rather better performance in semen assessment. Disposable chambers are suitable for routine semen analysis with CASA in a diagnostic andrology setting. With the proper workflow and quality control, a plain glass slide and the tissue culture dish cover are acceptable alternatives for routine counting chambers with CASA as necessary. The type of counting chamber should be specified in test reports.