Cyclic AMP-response element binding protein zhangfei (CREBZF), a member of ATF/CREB (activating transcription factor/ cAMP response element binding protein) family, regulates numerous cellular functions and development of cells by interacting transcription factors. This study discovered the expression pattern of CREBZF in seminiferous tubule of testes during the postnatal development of mice. In testis, CREBZF mRNA expression was the highest among other organs. Immunofluorescence analyses showed that the CREBZF was specifically expressed on spermatocyte but not in spermatogonia and Sertoli cells in seminiferous epithelium of mouse testis. Semi-quantitative polymerase chain reaction (PCR) analysis showed that CREBZF transcript level was significantly elevated during postnatal development of mouse testis. Confocal imaging analysis indicated that the protein expression of CREBZF in seminiferous tubule remained low until postnatal day (PD) 14, and was dramatically increased in PD 21. Interestingly, only one type of the spermatocyte expressed CREBZF specifically among SCP3-positive spermatocytes. Taken together, these results suggest that CREBZF may be novel putative marker of the spermatocyte and regulate meiosis during postnatal development of mice.

Spermatogonial stem cells (SSCs; also known as Asingle [As] spermatogonia in mice) divide to self-renew or to produce progenitor cells known as Apaired(Apr) spermatogonia in basal compartment of seminiferous tubules of mammalian testis. These characterized cells are the finally differentiated product of a developmental process referred to as “spermatogenesis.” In the development of SSCs it is critical to maintain a balance between self-renewal and differentiation. because an excess of either process will lead to infertility. these two processes are tightly controlled by intrinsic signals of SSCs and extrinsic signals from the microenvironment, known as the SSC niche. The SSC niche is formed by Sertoli cells, the only somatic cells found inside the seminiferous tubules. The WNT/β-catenin pathway is known to regulate Sertoli cell functions critical to their capacity to support spermatogenesis in the postnatal testis, but The mechanisms and factors of the pathway are not well known. We found a factor TLE3 (Transducin Like Enhancer Of Split 3). The transcriptional co-repressor TLE family is known to function as transcription co-repressors within the context of Wnt signaling by interacting with histone deacetylase HDAC2. We examined the expression level of TLE3 in various mouse tissues. As a result of RT-PCR, TLE3 showed significantly higher expression in testis than that in other tissues. Immunofluorescent analysis revealed that TLE3 and HDAC2 expression are differentially regulated in the mouse testis during postnatal development. In adult testis, TLE3 and HDAC2 were co-expressed in Sertoli cells. TLE3 and HDAC2 protein are also located in nucleus in mouse TM4 Sertoli cells. Taken together, TLE3 may play a role in regulating WNT/β-catenin pathway via interaction with HDAC2 in Sertoli cell. Futher studies are needed to look into factors that regulated by siTLE3 in Sertoli cell and interated with TLE3 in WNT/β-catenin pathway.

To overcome the risk of the ovarian hyperstimulation syndrome (OHSS) in patients have polycystic ovarian syndrome (PCOS) and to prepare emergency fertility preservation in patients undergoing anticancer treatment, several researchers have reported IVM of oocytes retrieved from ovaries exposed by only hCG priming. However, the maturation rate and the developmental potential of embryos from IVM oocytes are significantly lower than those of oocytes matured in vivo. Here, we investigated the optimal time point for immature oocyte collection at post hCG only injection for in vitro maturation, in vitro fertilization and blastocyst formation. Immature GV oocytes were collected from 25 days old B6D2F1 female mouse at 12 hr, 14 hr, 16 hr or 24 hr post hCG injection. Oocytes were collected from antral or late secondary follicle by puncturing with 26 G needle. Collected oocytes were cultured in G2 medium with 10% FBS, FSH, estradiol, and hCG for 16 hr in vitro and subjected in vitro fertilization and further embryonic development. To examine follicular maturation, we estimated the numbers of primordial, primary, secondary follicle and antral follicle on ovaries of each time point post hCG. To confirm the optimal time point post hCG injection for collecting immature oocytes, we recovered the oocytes from each time point. There is no difference in the number of oocytes per mice. Oocytes collected at 14 hr post hCG injection were shown higher maturation rate to MII stage and blastocyst formation compare to other three groups (p<0.01). However, there is no difference in the maturation rate on the other three groups. Also, apoptotic signal with TUNEL assay or anti-PARP staining was not change in ovaries from all experimental groups. Granulosa cell proliferation test with anti Ki-67 or anti AMH was not show any difference. According to these results, there are no significant differences in four different time points at 12 hr, 14 hr, 16 hr or 24 hr of collection of immature oocytes in hCG primed mouse. However, oocytes from 14 hr post hCG injection showed higher percentages of maturation rate, in vitro fertilization rate, blastocyst formation.

Cryopreservation has become a powerful method of the assisted reproduction technology and supports fertility preservation of cancer and other indication patients. After controlled ovarian hyperstimulation, surplus oocytes and embryos were recommended to store using cryopreservation. Recently, vitrification is replaced with traditional slow freezing protocol, because of improved survival rates and clinical outcomes. Vitrification requires a high concentration of CPAs that may induce significant osmotic and metabolic damage to cells including oocytes even in a short exposure of a few minutes. Generally, MPF plays a crucial role in the cell cycle regulation and maintaining the meiotic arrest of oocytes. In fact, it has been observed to decline in MII ovine oocytes after vitrification and would be suggested that one of the main causes of low fertilization rate and developmental competence derived from cryoinjury during vitrification. Therefore, the aim of this study was to evaluate the effect of caffeine treatment on the activity of MPF, MAPK level in vitrified/warmed mouse mature eggs. Caffeine, Phosphataseinhibitor, may maintain active form of MPF. We evaluated their survival after warming procedure, fertilization, cleavage, and developmental rates. Ovulated MII eggs were retrieved from 6 weeks old B6D2F1 female mouse at 14hr post hCG injection. Collected MII eggs were maintained in HTF medium containing 10% KSR with or without caffeine for 1hr. Eggs were vitrified in 7.5%EG +7.5%DMSO equilibrium solution, 15%EG + 15%DMSO + 0.5M sucrose vitrification solution with or without caffeine. Also warming solution contained sucrose (0.5M, 0.25M, 0.125M, and 0M) with or without caffeine. After warming, eggs were cultured in HTF medium with or without caffeine for 2 hr then fertilized with epididymal sperm in vitro and cultured in KSOM for 5 days to analyze embryonic development. Survival rates were similar in all experimental groups. However, fertilization rate was higher in with caffeine group compare to without caffeine significantly (80% vs. 85%, p<0.05). 2-cell and blastocyst formation were increased in caffeine group (p<0.05). MPF activity and MAP kinase activity were recovered in with caffeine group after vitrification/warming process. In conclusion, Caffeine may maintain MPF and MAPK level in vitrified/warmed MII eggs, and enhance fertilization and further embryonic development.

Estrogen is an important regulator of reproduction in both male and female. The two forms of estrogen receptor (ER) are known, ERα and ERβ. To understand the role of ERα in the testis, we investigated the expression of ERα in the mouse Leydig cells during postnatal development and the effects of estrogen on steroidogenesis and proliferation in progenitor Leydig cells (PLCs). In the testis, ERα mRNA and protein levels were markedly increased from postnatal day (PND) 1 to 14 and decreased thereafter until PND 56. During postnatal development ERα immunoreactivity was strong in the nucleus of Leydig cells at PND 14 when PLCs were abundant in the interstitium and low in the mature adult Leydig cells (ALCs). In fetal Leydig cells (FLCs), ERα immunoreactivity was negligible at birth and became increased at PND 14. This suggests an important role of ERα in Leydig cells during neonatal period. In isolated PLCs, 17β-estradiol (E2) and ERα-selective agonist, PPT suppressed the hCG-induced progesterone production and steroidogenic pathway genes expression. The hCG-induced PLCs proliferation was significantly inhibited by E2 and PPT. In conclusion, estrogen - ERα signaling may negatively regulate functional differentiation and proliferation of PLCs.

p63은 다양한 상피 조직의 줄기세포와 전구세포에 존재한다는 사실이 잘 알려져 있으나, 치아 형성, 특히 사기질과 뿌리 형성시기에서의 p63 위치느 ㄴ아직 연구해야 할 과제로 남아 있다. 본 연구에서는 p63이 치아 발생 동안 치아상피에 편재하여 나타나는 것을 면역조직화학 기법을 이용하여 확인하였다. p63은 피부, 모낭, 구강점막 그리고 턱밑샘 도관을 포함하는 상피의 바닥층과 바닥위층에 위치하였다. 그러나 치아 부위에서는 치아관의 모든 세포, 사기질기

내분비계 장애물질은 다양한 동물의 생식과 배아의 발생에 위해한 영향을 미친다고 보고되었다. 이러한 초기 배아의 비정상 발생 중 대표적인 것이 할구파편이다. 본 연구에서는 착상 전 생쥐 초기 배아를 PCB, BPA, DDT에 체내, 체외에서 각각 노출시켰다. 내분비계 장애 물질에 노출시켰던 배아는 할구파편, 불규칙한 할구와 깨짐, 일부 파괴된 투명대 배아 등의 다양한 형태학적 비정상 양상을 보였다. 착상 전 생쥐 초기배아의 세포괴사에 관여하는 유전자를 조

Insulin과 tumor necrosis factor alpha(TNF-)에 의한 초기 배아 발생의 조절기작을 알아보고자 생쥐의 상실배를 대상으로 이들이 첨가된 배양액에서 형태발생, 세포증식을 조사하고, 포배에서 mitogen activated protein kinase(MAPK, ERK1/2)의 활성 변화에 미치는 영향을 조사하였다. Insulin은 상실배의 체외발생 및 포배내 할구 수를 대조군에 비해 유의하게 증가시켰으며, TNF-는 발생율을 유의