• Development and Reproduction
• /
• v.1 no.2
• /
• pp.189-202
• /
• 1997

The hypothalamic peptide GnRH plays a central role in the regulation of the mammalian reproductive axis. Recent studies suggested that GnRH stimulates or inhibits the ovarian steroidogenesis and gametogenesis directly. Our previous report indicated that GnRH gene is expressed in adult rat ovary as well as in hypothalamus and that the expressed GnRH may induce the follicular atresia and apoptosis of ovarian granulosa cells in rat. Therfore, we studied whether GnRH gene is expressed in the mouse fetal ovary, when the germ cells are degenerating by apoptosis during gonad diffeerentiation. Mouse fetal gonads were obtained on the 12, 15,18 and 20th day of gestation from the mother mice superovulated (10 IU PMSG and 10 IU hCG) and mated. The morphological changes of fetal ovaries were examined histochemically by hematoxylin-eosin staining. The fetal sex was confirmed by PCR methods for sexing. RT-PCR methods were used to examine the expression of GnRH gene and the sex steroid hormones were determined by conventional radioimmunoassays. The levels of estradiol (E) and progesterone (P) were increaseduntil 18th day of gestation and then E was decreased just before parturition. The morphological changes of fetal gonadal tissue sections showed the ovarian development and coincided with the result of PCR analysis for sexing using ovary- or testis- specific oligonucleotide primers. Immunoreactive GnRH in placenta was decreased gradually until the end of gestation but fetal brain and ovarian GnRH were increased. The level of GnRH gene expression was increased during fetal ovarian development from 12 till 18th day and decreased suddenly on 20th day just before birth. From these results, it is suggested that ovarian GnRh may play a regulatory role on the germ cell differentiation of fetal ovary.

• Development and Reproduction
• /
• v.22 no.4
• /
• pp.331-339
• /
• 2018

NUCB2/nesfatin-1 is first known to be expressed in the hypothalamus while controlling appetite and energy metabolism. However, recent studies have shown that NUCB2/nesfatin-1 was expressed in the various organs as well as the hypothalamus. Our previous reports also demonstrated that NUCB2/nesfatin-1 was expressed in the ovary, testis, pituitary gland, lung, kidney, and stomach of fetal and adult mice. However, the role of NUCB2/nesfatin-1 in mouse fetus remains unknown. Thus, the aim of this study was to investigate whether NUCB2/nestatin-1 is expressed in mouse fetus at the developmental stage in which organogenesis begins. To do this, we performed in situ hybridization (ISH) and immunohistochemistry (IHC) staining to examine the distribution of NUCB2 mRNA and nesfatin-1 protein in the mouse fetal organs during early developmental stages, especially at embryonic day (E) 10.5. As a result of ISH, NUCB2 mRNA positive signals were more frequent in the liver, but there were relatively few positive signals in heart. On the other hand, no positive signals were detected in other organs. These ISH results were validated by IHC staining and qRT-PCR analysis. Expression of nesfatin-1 protein detected by IHC staining was similar to that of NUCB2 mRNA detected by ISH in the liver and heart. In addition, the levels of NUCB2 mRNA expression analyzed by qRT-PCR were significantly increased in the liver and heart compared to other organs of the mouse fetus at E13.5, whereas its level was extensively decreased in the liver, but increased in the lung, stomach, and kidney of the mouse fetus at E17.5. These results suggest that NUCB2/nesfatin-1 may play an important role in liver and heart development and physiological functions in the developmental process of mouse fetus. Further studies are needed on the function of NUCB2/nesfatin-1, which is highly expressed in the various organs, including liver and heart during mouse development.

• Development and Reproduction
• /
• v.2 no.1
• /
• pp.1-8
• /
• 1998

In mammalian ovary, major portion(>99%) of ovarian follicles undergo atresia. Recent studies have shown that this phenomenon is mediated via GC apoptosis. Ceramide, a product of sphingomyelin hydrolysis, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In the present study, we have examined the effect of ceramide on apoptotic cell death of GC in vitro. GCs were harvested by squeezing the antral follicles from the immature mice (3-4 weeks) and cultured in MEM medium with 10% fetal bovine serum. The cells were treated with various concentrations of ceramide (0 to 50 \mu M)and cultured up to 24 h.Cell death was determined by MTT cell viability assay and apoptosis was examined by acridine orange staining, in situ 3'-end labeling(TUNEL), and flow cytometry. Ceramid treatment induced apoptotic cell death of GC in a time- and a dose-dependent manner. Results of flow cytometric analysis showed that creamide-induced cell death was mostly confined to the $G_{0}$/$G_{1}$ cells. these results provide an evidence for ceramide as a lipid second messenger of apoptosis in mouse GC.

• Development and Reproduction
• /
• v.3 no.1
• /
• pp.101-105
• /
• 1999

N-Methylpurine-DNA glycosylase (MPG) removes N-methylpurine and other damaged purines in DNA. RT-PCR analysis revealed MPG mRNA expression at various tissues of fetal development from day 8 to day 18 fetus and day 400 mature adult. The MPG transcripts were abundant during fetal development in mice. In placenta, the MPG mRNA was continuously decreased from day 8 post coitum (p.c) to day 18 p.c. fetus. The high level of mRNA in fetal brain and liver was drastically declined in day 400 mature adult. The expression of MPG, originally characterized by its highest level of expression in the epididymis of adult mouse, was detected with high level in several other reproductive organ, including the ovary, oviduct, testis, vas deference, uterus, and seminal vesicles. These results demonstrate developmental stage- and tissue-specific variation of MPG gene expression.

• Development and Reproduction
• /
• v.3 no.1
• /
• pp.95-100
• /
• 1999

Growth differentiation factor-9 (GDF-9) is a member of the transforming growth factor $\beta$ (TGF-$\beta$) superfamily. It has been known that GDF-9 is a growth factor having a crucial role in normal folliculogenesis and its expression is oocyte-specific. The present study was aimed to elucidate the expression of GDF-9 mRNA in the mouse primordial follicles as well as in the other developmental stages. The semiquantitative analysis of GDF-9 mRNA expression was conducted. Total RNA was extracted from the ICR mice ovaries at gestational day 19, postnatal day 1, day 10, day 21, and day 28, and RT-PCR was performed to measure GDF-9 and $\beta$-actin mRNA levels. Level of GDF-9 mRNA were normalized against the level of $\beta$-actin mRNA, and compared among different stages. GDF-9 mRNA was detected in all samples including the fetal ovaries that mainly consists of primordial follicles. The highest level of mRNA was observed in ovaries obtained at day 10 that mainly consists of growing follicles. The present result suggests that GDF-9 may play an important role in the early stage of folliculogenesis.

• Journal of Microbiology and Biotechnology
• /
• v.25 no.12
• /
• pp.1989-1996
• /
• 2015

Ice-binding proteins (IBPs) can inhibit ice recrystallization (IR), a major cause of cell death during cryopreservation. IBPs are hypothesized to improve cell viability after cryopreservation by alleviating the cryoinjury caused by IR. In our previous studies, we showed that supplementation of the freezing medium with the recombinant IBP of the Arctic yeast Glaciozyma sp. (designated as LeIBP) could reduce post-thaw hemolysis of human red blood cells and increase the survival of cryopreserved diatoms. Here, we showed that LeIBP could improve the viability of cryopreserved mammalian cells. Human cervical cancer cells (HeLa), mouse fibroblasts (NIH/3T3), human preosteoblasts (MC3T3-E1), Chinese hamster ovary cells (CHO-K1), and human keratinocytes (HaCaT) were evaluated. These mammalian cells were frozen in dimethyl sulfoxide (DMSO)/fetal bovine serum (FBS) solution with or without 0.1 mg/ml LeIBP at a cooling rate of -1℃/min in a -80℃ freezer overnight. The minimum effective concentration (0.1 mg/ml) of LeIBP was determined, based on the viability of HeLa cells after treatment with LeIBP during cryopreservation and the IR inhibition assay results. The post-thaw viability of mammalian cells was examined. In all cases, cell viability was significantly enhanced by more than 10% by LeIBP supplementation in 5% DMSO/5% FBS: viability increased by 20% for HeLa cells, 28% for NIH/3T3 cells, 21% for MC3T3-E1, 10% for CHO-K1, and 20% for HaCaT. Furthermore, addition of LeIBP reduced the concentrations of toxic DMSO and FBS down to 5%. Therefore, we demonstrated that LeIBP can increase the viability of cryopreserved mammalian cells by inhibiting IR.

• Clinical and Experimental Reproductive Medicine
• /
• v.27 no.3
• /
• pp.235-243
• /
• 2000

Objective: Our present studies were conducted to examine more effective isolating method of preantral follicles from mouse ovaries. Methods: ICR mice (3-6 weeks old) were sacrificed through cervical dislocation and their ovaries were removed and put into watch glasses containing Hams F-10 supplemented with 10% fetal bovine serum (FBS). Preantral follicles were isolated by three different methods; 1) enzymatical method and 2) mincing method, and 3) scraping method. Enzymatical method was carried out as following. Ovaries were bisected with a pair of fine 30G needles. Bisected ovaries were incubated at $37^{\circ}C$ and 5% $CO_2$ incubator in 2-well dish containing Hams F-10 supplemented with collagenase 600 lU/ml and DNAse 20 lU/ml. After 20 min., follicles were isolated by repeated pipetting. Isolated preantral follicles were collected, and the remnant of tissues was placed in incubator and previous procedure was repeated. Mincing method was carried out with a pair of fine 30G needles attached to 1 ml syringes and minced ovary. Scraping method was carried out with a pair of fine 30G needles and scratched to surface of ovary. The differences between isolating methods were analyzed using Student's t-test and Chi-square. Results were considered statistically significant when ${\rho}$ value was less than 0.05. Results: In handling time, mincing or scraping method ($28{\pm}3.42$ min or $16{\pm}1.58$ min) were significantly (p<0.00001) shorter than enzymatical method ($72{\pm}1.69$ min), and scraping method was significantly (p<0.01) shorter than mincing method. Total number of isolated follicles was significantly (p<0.0001) higher in enzymatical method ($49.8{\pm}3.91$) than in mincing or scraping method ($25.3{\pm}2.33$ or $20.5{\pm}1.75$). Isolated follicles in ${\leq}$90${\mu}m$ were significantly (p<0.005) higher in enzymatical method ($15{\pm}1.71$) than in mincing or scraping method ($7.8{\pm}0.98$ or $8.1{\pm}1.31$). In 91~130 ${\mu}m$, isolated follicles were significantly (p<0.0005) higher in enzymatical method ($33{\pm}3.27$) than in mincing or scraping method ($16.3{\pm}1.82$ or $10.7{\pm}1.38$). In ${\geq}$ 131 ${\mu}m$, isolated follicles were not significantly differences between all groups. In equal sizes, the rate of isolated follicles in ${\leq}$ 90 ${\mu}m$ was highest in scraping method (39.6% vs. enzymatical method: 30.1%, p<0.05; mincing method: 30.9%, p=0.11719, NS). Rate of follicles in $91{\sim}130$ ${\mu}m$ was significantly (p<0.05) lower in scraping method (52.7%) than in enzymatical or mincing method (66.3% or 64.5%). Rate of follicles in ${\geq}$131 ${\mu}m$ was highest in scraping method (8.3% vs. enzymatical or scraping method: 3.6%, p<0.05 or 4.6%, p=0.19053, NS). Conclusions: This study suggests that scraping method is simple and useful for isolation of preantral follicles, because this method reduced handling time and recovered enough follicles. The recovered rate of isolated follicles in diameter of 91 ~ 130 ${\mu}m$ was highest in all methods.