Differential capacity of the parthenogenetic embryonic stem cells (PESCs) is still under controversy and the mechanisms of its neural induction are yet poorly understood. Here we demonstrated neural lineage induction of PESCs by addition of insulin-like growth factor-2 (Igf2), which is an important factor for embryo organ development and a paternally expressed imprinting gene. Murine PESCs were aggregated to embryoid bodies (EBs) by suspension culture under the leukemia inhibitory factor-free condition for 4 days. To test the effect of exogenous Igf2, 30 ng/ml of Igf2 was supplemented to EBs induction medium. Then neural induction was carried out with serum-free medium containing insulin, transferrin, selenium, and fibronectin complex (ITSFn) for 12 days. Normal murine embryonic stem cells derived from fertilized embryos (ESCs) were used as the control group. Neural potential of differentiated PESCs and ESCs were analyzed by immunofluorescent labeling and real-time PCR assay (Nestin, neural progenitor marker; Tuj1, neuronal cell marker; GFAP, glial cell marker). The differentiated cells from both ESC and PESC showed heterogeneous population of Nestin, Tuj1, and GFAP positive cells. In terms of the level of gene expression, PESC showed 4 times higher level of GFAP expression than ESCs. After exposure to Igf2, the expression level of GFAP decreased both in derivatives of PESCs and ESCs. Interestingly, the expression level of $Tuj1$ increased only in ESCs, not in PESCs. The results show that IGF2 is a positive effector for suppressing over-expressed glial differentiation during neural induction of PESCs and for promoting neuronal differentiation of ESCs, while exogenous Igf2 could not accelerate the neuronal differentiation of PESCs. Although exogenous Igf2 promotes neuronal differentiation of normal ESCs, expression of endogenous $Igf2$ may be critical for initiating neuronal differentiation of pluripotent stem cells. The findings may contribute to understanding of the relationship between imprinting mechanism and neural differentiation and its application to neural tissue repair in the future.
Purpose : These studies were undertaken to evaluate the effects of the Cynomorii Herba (CH) on the spermatogenic abilities such as the concentration, motility and morphological normality of sperm from the testis, and the activities of sperm hyaluronidase, testicular peroxidase and testicular catalase. Materials and Methods : We used the 2-month-old mice and administered 0.2ml extract solution of CH in the 0.1mg/ml, 1mg/ml, 10mg/ml and 100mg/ml once a day for 60days. The control group was administered the distilled water in the same way. After the administration of extract solution, we examined the number of total, motile and normal sperm from the cauda epididymis, the activities of sperm hyaluronidase, testicular peroxidase and testicular catalase. We observed the histological changes of isolated testis and compared to the testicular tissue especially seminiferous tubules between control and CH groups by histochemical method. Results : The concentration of total sperm and the motility of spermatozoa were significantly increased in the 1mg/ml, 10mg/ml and 100mg/ml CH groups, especially in 10mg/ml group, compared to the control group. The significant differences were observed in the normality of spermatozoa of the CH groups compared to the control group. In the histolocal analysis of the testicular tissues, the enlargement of testicular lobe diameter and apparent vasculogenesis between testicular lobes were observed in the CH groups compared to the control group. Also, the activity of hyaluronidase was significantly increased in the CH groups compared to the control group. In the antioxidant activity analysis, the activities of testicular peroxidase and testicular catalase were significantly increased in the CH groups compared to the control group, respectively. Conclusion : This study shows that CH has the beneficial effect on the concentration, morphology and motility of sperm, the activities of sperm hyaluronidase, testicular peroxidase and testicular catalase. We can suggest that CH extract solution be useful for the treatment of male sexual dysfunctions and infertility.
Purpose: This study was undertaken to evaluate the effects of the different administration duration of Cynomorii Herba extract solution on the spermatogenic abilities such as concentration, motility and morphological normality of sperm from the testis and the activities of sperm hyaluronidase. Methods : We used the 8-week-old ICR mice and administered 0.3mg/g extract solution of Cynomorii Herba once a day for 30, 60, 90 and 120 days. The control group was administered the normal saline in the same way and duration. We examined the number of total, motile and normal sperm from the cauda epididymis. And we compared the testicular tissue especially seminiferous tubules between control and treated groups by histochemical methods. At the end we observed the difference of sperm hyaluronidase activities between control and treated groups. Results : The significant differences were observed in the concentration of total sperm, the motility and normality of spermatozoa of the Cynomorii Herba extract solution administered groups compared to the control group in 60 and 90 days groups. In the histological analysis of the testicular tissues, the enlargement of testicular lobe diameter and apparent vasculogenesis between testicular lobes were observed in the Cynomorii Herba extract solution administered groups compared to the control group, respectively. Also, the activity of hyaluronidase was significantly increased in the Cynomorii Herba extract solution administered groups compared to the control group. Conclusion : This study shows that Cynomorii Herba has the beneficial effect on the concentration, morphology and motility of sperm especially in 60 and 90 days administration group. We can suggest that Cynomorii Herba extract solution be useful for the treatment of male sexual dysfunctions and infertility.
Purpose : These studies were undertaken to evaluate the effects of the administration of different concentrated Morindae officinalis Radix extract solution on the spermatogenic abilities such as concentration, motility and morphological normality of sperm from the testis and the activities of sperm hyaluronidase, testicular peroxidase and testicular catalase. Methods : We used the 2-month-old mice and administered the extract solution of Morindae officinalis Radix in the different concentration once a day for 60 days. The control group was administered the normal saline in the same way and duration. We examined the number of total, motile and normal sperm from the cauda epididymis, the activities of sperm hyaluronidase, testicular peroxidase and testicular catalase. Also we observed changes of isolated testis before and after administration of Morindae officinalis Radix extract solutions the mice. And we compared to the testicular tissue especially seminiferous tubules between control and treated group by histochemical methods. Results : The significant dose-dependent differences were observed in the concentration of total sperm, the motility and normality of spermatozoa of Morindae officinalis Radix extract solution administered groups compared to the control group, respectively. In the histological analysis of the testicular tissues, the enlargement of testicular lobe diameter and apparent vasculogenesis between testicular lobes were observed in the Morindae officinalis Radix extract solution administered groups compared to the control group, respectively. Also, the activity of hyaluronidase was significantly increased in the Morindae officinalis Radix extract solution administered groups compared to the control group. In the antioxidant activity analysis, the activity of testicular peroxidase was significantly increased in the Morindae officinalis Radix extract solution administered groups compared to the control group, respectively. Conclusion : This study shows that Morindae officinalis Radix has the beneficial effect on the concentration, morphology and motility of sperm, the activities of sperm hyaluronidase and testicular peroxidase. We can suggest that Morindae officinalis Radix extract solution be useful for the treatment of male sexual dysfunctions and infertility.
The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.
This study was carried out to investigate the optimal culture conditions for gametophytes growth and sporophytes regeneration of Pyrrosia linearifolia in order to provide for the masspropagation system foundation of Pyrrosia linearifolia using their life cycle. Among many different media, 2MS medium was most effective in prothallus proliferation. Prothallus growth was promoted as the total concentration of nitrogen sources increased, and the best result was observed on 120 mM nitrogen. The best concentration of sucrose was 3%. The addition of 5~20 mM IAA, NAA, BA and kinetin promoted the propagation of prothallus. But 2iP demonstrated the most inhibitory effect on prothallus proliferation. Gametophytes shaking-cultured with liquid medium showed similar growth with solid medium and normal formation of reproductive organs. Shoot regeneration was most effective on 1/8MS medium, but growth was promoted on 1/2MS medium. For promotion of shoot regeneration and growth, the suitable concentrations of sucrose and $NaH_2PO_4$ were 1% and $50{\sim}100mg{\cdot}mL^{-1}$ in 1/8MS medium, respectively.
Leptin, the product of obese (ob) gene, is an adipocyte-derived satiety factor that plays a major role in the regulation of food intake, energy homeostasis, body weight, reproductive physiology and neuropeptide secretion. The present study was designed to generate transgenic mice expressing antisense mouse ob (mob) gene. Total RNA was extracted from the adipose tissues of mouse, then reverse transcription was performed. The 303 and 635 bp fragments of anti I and II cDNAs were amplified from mob cDNAs by PCR. The two mob cDNAs were reversely ligated into between adipose tissue specific aP2 promote and SV40 poly(A) site. Transgenic mice carrying two different kinds of antisense mob transgenes were generated by DNA microinjection into pronucleus. Total 14 transgenic mice were born, and the 4 and 5 founder lines of the transgenic mice with anti I and II transgenes were respectively established. Antisense mRNA expression was detected in transgenic F$_1$ mice by RT-PCR analysis. This result suggests that the transgenic mice expressing antisense mob mRNA may be useful as an animal disease model to be obesity caused by decreased amount of leptin secretion.
Ethane 1,2-dimethane sulfonate (EDS) is a well-known alkylating agent used as selective Leydig cell (LC) toxicant to create a testicular dysfunction model. Previous studies including our own clearly demonstrated the dramatic weight loss of the androgen dependent accessory sex organs such as epididymis, seminal vesicle and prostate gland in this 'LC knock-out' rats. The present study was performed to evaluate the effect of EDS administration on histological changes of the epididymis, seminal vesicle and prostate in adult rats. Adult male Sprague-Dawley rats (350$\sim$400 g B.W.) were injected with a single dose of EDS (75 mg/kg, i.p.) and sacrificed on weeks 0, 1, 2, 3, 4, 5, 6 and 7. Tissue weights (testis, epididymis, seminal vesicle and prostate gland) were measured. The histological changes of tissue were observed by a light microscopy using hematoxylin & eosin staining. Weights of the reproductive and accessory organs progressively declined after the EDS treatments (weeks 1, 2 and 3). After this, the decrease was stopped, then gradually returned to the normal levels. There was a partial (about 60%) recovery of the epididymis weight during weeks $6{\sim}7$. The cross section of epididymis revealed an increase in thickness of the epithelium during weeks $1{\sim}3$. In contrast, considerable reduction of epithelial thickness in seminal vesicle was observed during same period. Similarly, a reduction in thickness of prostate epithelial layer was found during weeks $1{\sim}3$, then it was back to normal thickness after week 4. Taken together, the present study demonstrated that the temporally induced androgen-deficiency by EDS treatment could result the prominent alterations in histology of the accessory sex organs. Further studies on the physiological and molecular regulation of these androgen-sensitive organs using EDS model will be helpful to understand the normal and pathological development and differentiation mechanism of these organs.
Polycystic ovary syndrome (PCOS) is hormonal imbalance condition as the endocrine and metabolic disorder that induces the infertility and various complications in reproductive age women. Estradiol valerate (EV) is used hormone replacement therapy in menopausal women and is reported that excessive administration of EV induces the PCOS. Nerve growth factor (NGF) is the factor to regulate the survival and maturation of developing neuronal cell and is also synthesized in ovary. And NGF is overexpressed in EV-induced polycystic ovary (PCO) as previously reported. Therefore, this study examined the possibility of NGF as can be used the biological marker in diagnosis of PCOS, the hormonal imbalance condition, using PCO and CHO (chinese hamster ovarian) cell lines. The concentration of EV treatment is optimized a 1 mg as not influence on the proliferation of CHO cell but 2 mg and 3 mg of EV treatment have the inhibition effect at initial stage. The morphological change was not observed in CHO cell after dose dependent manner treatment of EV. Expression of NGF mRNA and protein is significantly increased at 30 min after EV treatment in CHO cells compared to that of control. And NGF protein expression is strongly increased in PCO tissue, which observed many follicular cysts compared to normal ovary tissue. Taken together, overexpression of NGF may be act as a molecule to induce an abnormal development of follicle, suggesting that NGF can be used as a biological marker in diagnosis of PCOS.
The present study was carried out to examine the effect of four different media (NCSU (North Carolina State University)-23, PZM (Porcine Zygotes Medium)-3, PZM-4 and TCM (Tissue Culture Medium)-l99) and two oxygen concentrations (39 , 5% $O_2$, 5% $CO_2$ and 90% $N_2$, 5% $CO_2$ in air) on in vitro production of porcine IVM/IVF embryos. The results were summarized as follows: The rates of GVBD and nuclear maturations were not significantly different (p>0.05) for 44 hours of culture with four media in two oxygen concentrations. The rates of polyspermy, penetrated sperm(s) and male and female prouclei formation were not significantly different (p>0.05). among four media in two oxygen concentrations. The cleavage rates were not significantly different (p>0.05) among four media in two oxygen concentrations. At day 7 under gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$, the blastocyst formation was significantly higher (p<0.05) in PZM-3 (19.9$\pm$2.4) than other media. Also, NCSU-23 medium gave high rate of blastocyst formation at day 7 under gas atmosphere of 5% $CO_2$ in air (p<0.05). Based on the result of differential staining of porcine blastocyst at dat 7, inner cell mass cell and total cell numbers were not significantly different (p>0.05) among four media in two oxygen concentrations. However, the observed total cell number was higher in PZM-3 medium (36.8$\pm$6.5) than other madia. In conclusion, these results suggested that in vitro production of porcine embryos in PZM-3 medium under a gas atmosphere of 5% $O_2$, 5% $CO_2$ and 90% $N_2$ was effective on the blastocyst formation rate and total blastocyst cell number.
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