Polyploidy is occurred by the process of endomitosis or cell fusion and usually represent terminally differentiated stage. Their effects on the developmental process were mainly investigated in the amphibian and fishes, and only observed in some rodents as mammalian model. Recently, we have established tetraploidy somatic cell nuclear transfer-derived human embryonic stem cells (SCNT-hESCs) and examined whether it could be available as a research model for the polyploidy cells existed in the human tissues. Two tetraploid hESC lines were artificially acquired by reintroduction of remained 1st polar body during the establishment of SCNT-hESC using MII oocytes obtained from female donors and dermal fibroblasts (DFB) from a 35-year-old adult male. These tetraploid SCNT-hESC lines (CHA-NT1 and CHA-NT3) were identified by the cytogenetic genotyping (91, XXXY,-6, t[2:6] / 92,XXXY,-12,+20) and have shown of indefinite proliferation, but slow speed when compared to euploid SCNT-hESCs. Using the eight Short Tendem Repeat (STR) markers, it was confirmed that both CHA-NT1 and CHA-NT3 lines contain both nuclear and oocyte donor genotypes. These hESCs expressed pluripotency markers and their embryoid bodies (EB) also expressed markers of the three embryonic germ layers and formed teratoma after transplantation into immune deficient mice. This study showed that tetraploidy does not affect the activities of proliferation and differentiation in SCNT-hESC. Therefore, tetraploid hESC lines established after SCNT procedure could be differentiated into various types of cells and could be an useful model for the study of the polyploidy cells in the tissues.

BACKGROUND Ca2+ oscillations during fertilization induce eggs activation and embryonic development in mammalian eggs.. The type 1 inositol 1,4,5-trisphosphate receptor (IP3R1) is in charge of Ca2+ oscillations for the release of stored Ca2+ from the endoplasmic reticulum. The capacity of this oscillation is obtained during egg maturation and corresponds with an increase in the sensitivity of the IP3R1 and their localization in cytoplasm. Cluster formation of IP3R1 in the egg cortex is important to initiation of Ca2+ oscillations during egg and sperm fusion. In this study, we investigated that cell cycle–coupled redistribution of IP3R1 and Ca2+- oscillatory activity in mouse zygotes. MATERIALS AND METHODS Metaphase II arrested eggs were collected from ICR female mouse after super ovulation induction. At 14 hr post hCG, MII eggs were collected, and artificially activated in Ca2+ free CZB medium with 10 mM SrCl2 for 2 hrs. Pronuclear zygotes (PN) were collected from Strontium activated eggs at 8 hr post activation, and the first mitotic eggs were collected at 16~17 hr post activation. To identify cell cycle coupled IP3R1 redistribution, MII eggs, zygotes, and first mitotic eggs were collected, and fixed for immunostaining with anti-IP3R1antibody (CT-1) and observed on CLSM. Ca2+-oscillatory activity was monitored with fluorescence microscope mounted SimplePCI program (Hamamatsu) after injection of cRNA of mouse phospholipase C zeta (mPLCZ). RESULT IP3R1 were shown clusters, 1~2 um in diameter, in cortex of ovulated MII eggs with high Ca2+ oscillatory activity by mPLCZ injection. These eggs represent more than 6 spikes per 60 min. However, IP3R1 clusters were disappeared in PN eggs and these eggs showed very low Ca2+- oscillatory activity by mPLCZ. In mitosis I stage eggs, clusters of IP3R1 were appeared and Ca2+-oscillatory activity was reactivated slightly (2 spikes per 60 min). CONCLUSIOINS This study introduced the redistribution of IP3R1 clusters were occurred in egg activation according to cell cycle dependent manner. Also, functional modification of IP3R1 including protein phosphorylation was associated with cortical clustering of IP3R1 in cell cycle coupled Ca2+ oscillatory activity.

Mature mammalian oocytes are ovulated at the metaphase II stage of meiosis and complete the cell cycle by fertilization with sperm. During fertilization sperm release an egg activation protein, pho-spholipase C zeta (PLCZ) into oocyte cytoplasm, PLCZ hydrolyze PIP2 into IP3 and DAG. The elevation of IP3 concentration induces Ca2+ release from endoplasmic reticulum (ER) by binding to the IP3 receptor (IP3R) on the membrane of ER. Recent studies have shown that sperm from patients lacking expression of PLCZ1 or expressing mutant forms of PLCZ1 fail to induce [Ca2+]i oscillations or oocyte activation. Purified recombinant human PLCZ1 (hPLCZ1) protein evaluated its [Ca2+]i oscillation activity in mouse and human oocytes. Here we investigated that produced mouse PLCZ-specific antibodyrecognized the PLCZ protein in mouse testes. PLCZ antibody was raised in rabbits against 19-mer sequence at the C-terminus (MENKWFLSMVRDDFKGGKI) of mouse PLCZ protein. Sperm were fixed in 3.7% paraformaldehyde followed by permeabilization. Sperm were incubated in 5% normal goat serum (NGS) and then incubated overnight with anti-mouse PLCZ. Peanut agglutinin (PNA)-lectin was used for detection of the acrosome. Mouse testes from 6~8 weeks old ICR mouse were fixed in 10% formalinand serial sectioned at 5~8um. Testes tissues were immunostained with anti PLCZ antibody and peanut agglutinin(PNA) for acrosome staining. Produced anti mouse PLCZ antibody recognized 74 kDa protein in western and PLCZ is localized to the post-acrosomal region of mouse sperm and to the equatorial region of bull sperm. Mouse PLCZ protein wasdetected on spermatocytes, spermatid, but not on spermatogonia in seminiferous tubules. Some residual bodies on sperm neck and tail showed strong signal of PLCZ, but this staining was still present with antigenic peptide pretreatment to reduce non specific antibody reaction. Also this antibody reacted with the apical region (arrowheads) of principal cells, where secretory vesicles accumulate on the epididymal tissue. But antigenic peptide pretreatment did not remove this apical region staining. This study presents PLCZ protein is localized on the post-acrosomal region or equatorial region of mouse and bull sperm head. Also PLCZ protein in mouse testes expressed from spermatocytes to mature sperm on later stage of spermatogenesis.