• Development and Reproduction
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• v.6 no.1
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• pp.55-66
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• 2002

Embryonic stem cells(ES cells) are derived from the inner cell mass(ICM) of blastocysts, which have the potentials to remain undifferentiated, to proliferate indefinitely in vitro, to differentiate into the derivates of three embryonic germ layers. ES cells are an attractive model system for studying the initial developmental decisions and their molecular mechanisms during embryogenesis. Additionally, ES cells of significant interest to those characterizing the various gene functions utilizing transgenic and gene targeting techniques. We investigated the effects of reproductive hormones, gonadotropins(GTH) and steroids on the induction of differentiation and expressions of their receptor genes using the newly established mouse ES cells. We collected the matured blastocysts of inbred mice C57BL/6J after superovulation and co-cultured with mitotically inactivated STO feeder cells. After 5 passages, we confirmed the expression alkaline phosphatase(Alk P) activity and SSEA-1, 3, 4 expressions. The protocol devised for inducing ES differentiation consisted of an aggregation steps, after 5 days as EBs in hormone treatments(FSH, LH, E$_2$, P$_4$, T) that allows complex signaling to occur between the cells and a dissociation step, induced differentiation through attachment culture during 7 days in hormone treatments. Hormone receptors were not increased in dose-dependent manner. All hormone receptors in ES cells treated reproductive hormones were expressed lower than those of undifferentiated ES cell except for LHR expression in E$_2$-treated ES cells group. After hormone induced differentiation, at least some of the cells are not terminally differentiated, as is evident from the expression of Oct-4, a marker of undifferentiated. To assess their differentiation by gene expression, we analyzed the expression of 7 tissue-specific markers from all three germ layers. Most of hormone-treated group increased in the expression of gata-4 and $\alpha$ -fetoprotein, suggesting reproductive hormone allowed or induced differentiation of endoderm.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.1
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• pp.81-85
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• 1992

The effects of pulsatile GnRH therapy have been studied for the treatment of different forms of hypogonadotropic hypogonadism, including idiopathic hypogonadotropic hypogonadism and hypogonadism developed as a result of treatment with combination of surgery and irradiation for pituitary or hypothalamic tumor. GnRH was administered subcutaneously in a dose of $10{\mu}g$ every 2 hours with the pulsatile infusion pump. With GnRH therapy, all patients improved secretion of LH, FSH and testosterone. Testicular volumes increased. Spermatogenesis was induced in 8 patients. Pulsatile GnRH therapy is an effective treatment for idiopathic hypogonadotropic hypogonadism and can have a role in hypogonadism previously treated with combination of surgery and irradiation for pituitary or hypothalamic tumor.

• Development and Reproduction
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• v.14 no.1
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• pp.35-41
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• 2010

Mammalian reproduction is regulated by a feedback circuit of the key reproductive hormones such as GnRH, gonadotropin and sex steroids on the hypothalamic-pituitary-gonadal axis. In particular, the onset of female puberty is triggered by gain of a pulsatile pattern and increment of GnRH secretion from hypothalamus. Previous studies including our own clearly demonstrated that genistein (GS), a phytoestrogenic isoflavone, altered the timing of puberty onset in female rats. However, the brain-specific actions of GS in female rats has not been explored yet. The present study was performed to examine the changes in the activities of GnRH neurons and their neural circuits by GS in female rats. Concerning the drug delivery route, intracerebroventricular (ICV) injection technique was employed to eliminate the unwanted actions on the extrabrain tissues which can be occurred if the testing drug is systemically administered. Adult female rats (PND 100, 210-230 g BW) were anaesthetized, treated with single dose of GS ($3.4{\mu}g$/animal), and sacrificed at 3 hrs post-injection. To determine the transcriptional changes of reproductive hormone-related genes in hypothalamus, total RNAs were extracted and applied to the semi-quantitative reverse transcription polymerase chain reaction (RT-PCR). ICV infusion of GS significantly raised the transcriptional activities of enhanced at puberty1 (EAP-1, p<0.05), glutamic acid decarboxylase (GAD67, p<0.01) which are known to modulate GnRH secretion in the hypothalamus. However, GS infusion could not change the mRNA level of nitric oxide synthase 2 (NOS-2). GS administration significantly increased the mRNA levels of KiSS-1 (p<0.001), GPR54 (p<0.001), and GnRH (p<0.01) in the hypothalami, but decreased the mRNA levels of LH-$\beta$ (p<0.01) and FSH-$\beta$ (p<0.05) in the pituitaries. Taken together, the present study indicated that the acute exposure to GS could directly activate the hypothalamic GnRH modulating system, suggesting the GS's disrupting effects such as the early onset of puberty in immature female rats might be derived from premature activation of key reproduction related genes in hypothalamus-pituitary neuroendocrine circuit.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.4
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• pp.176-181
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• 2012

Objective: In 2009 anti-M$\ddot{u}$llerian hormone (AMH) assay was approved for clinical use in Korea. This study was performed to determine the reference values of AMH for predicting ovarian response to controlled ovarian hyperstimulation (COH) using the clinical assay data. Methods: One hundred sixty-two women who underwent COH cycles were included in this study. We collected data on age, basal AMH and FSH levels, total dose of gonadotropins, stimulation duration, and numbers of oocytes retrieved and fertilized. Blood samples were obtained on cycle day 3 before gonadotropin administration started. Serum AMH levels were measured at a centralized clinical laboratory center. The correlation between the AMH level and COH outcomes and cut-off values for poor and high response after COH was analyzed. Results: Concentration of AMH was significantly correlated with the number of oocytes retrieved (OPU; r=0.700, p<0.001). The mean${\pm}$SE serum AMH levels for poor ($OPU{\leq}3$), normal ($4{\leq}OPU{\leq}19$), and high ($OPU{\geq}20$) response were $0.94{\pm}0.15$ ng/mL, $2.79{\pm}0.21$ ng/mL, and $6.94{\pm}0.90$ ng/mL, respectively. The cut-off level, sensitivity and specificity for poor and high response were 1.08 ng/mL, 85.8%, and 78.6%; and 3.57 ng/mL, 94.4%, and 83.3%, respectively. Conclusion: Our data present clinical reference values of the serum AMH level for ovarian response in Korean women. The serum AMH level could be a clinically useful predictor of ovarian response to COH.

• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.131-140
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• 2001

Objective: To know the effects of xenoestrogen on spermatogenesis, we investigated the expression of cytochrome P450s enzymes (CYPscc, $CYP_{17{\alpha}}$, CYP19) and $3{\beta}$-HSD genes involved in steroidogenesis. Methods: Mouse testicular cells were prepared from 15-day-old ICR mice which had only pre-meiotic germ cells by enzyme digestion using collagenase and trypsin. Testicular cells were cultured in DMEM supplemented with FSH (0.1 IU/ml) and 10% FBS or medium with estrogen ($E_2$), bisphenol-A (BPA), octylphenol (OP; $10^{-9},\;10^{-7},\;10^{-6},\;10^{-5},\;10^{-4}M$, respectively) and aroclor 1254 (A1254) known as PCBs for 48 hours. The gene expression of cytochrome P450 enzymes were examined by semi-quantitive RT-PCR. The production of estrogen and testosterone was examined by RIA. Results: As results, expression of CYPscc mRNA was not significantly decreased, but $3{\beta}$-HSD and $CYP_{17{\alpha}}$. mRNA were significantly dose-dependent decreased. And production of testosterone and estrogen were not different except BPA and OP group ($10^{-5}M$). Conclusion: BPA, OP and A1254 might inhibit steroidogenesis by decreasing CYPscc, $3{\beta}$-HSD and $CYP_{17{\alpha}}$. mRNA expression in the mouse testis. These results suggest that BPA, OP and PCBs like as an endocrine disruptors inhibit the productions of steroidogenic enzymes and decrease the production of T and E by negative feedback mechanism. Therefore, these might disrupt steroidogenesis in Leydig cells of testis and would disturb testicular function and subsequently impair spermatogenesis.

• Journal of Environmental Health Sciences
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• v.33 no.5
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• pp.379-385
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• 2007

This study was aimed to investigate the estrogenic activity of Di-(2-ethylhexyl) adipate (DEHA) using immatured type uterotrophic assay. SD rats were treated with DEHA (40, 200, 1000mg/kg/day), estradiol-3-benzoate (EB) $(1{\mu}g/kg/day)$ as positive control on the assay. In immatured-type uterotrophic assay, relative organ weights of kidney and reproductive organs such as ovary at high-dose group were significantly increased compared to those of vehicle control group. DEHA did not influence the levels of serum FSH and LH, and uterine morphological changes such as luminal epithelial height, myometrial thickness and numbers of uterine gland, and BrdU indices. In these results, there was no significant variation by DEHA treatment, suggesting that DEHA appears not to be a endocrine disrupter with estrogenic activity.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.3
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• pp.241-245
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• 1994

Despite increasing success rate of IVF, poor response to ovarian stimulation remains a problem. So, attempts to improve ovarian responses, for example, by using combined gonadotropin-releasing hormone analogue(GnRH-a) and human menopausal gonadotropin(hMG) have shown limited success. It is reported that response of granulosa cells in vitro to FSH is stimulated by co-incubation with IGF-l, and IGF-l production can be increased by growth hormone. This suggest that combination regimen of G.H. and hMG may augment follicle recruitment. In fifteen patients who had previous history of poor ovarian response to gonadotropin stimulation after pituitary suppression with mid -luteal GnRH-a, the effectiveness of cotreatment with G.H. in IVF program was evaluated using a combination regimen of G.R. and hMG at Korea University Hospital IVF Clinic. Ovarian responses to gonadotropin stimulation in control and GH-treated cycles assessed by total dose and duration of hMG treatment, follicular development and peak $E_2$ level, number of eggs retrieved, and fertilization rates were also assessed. In each group, serum and follicular fluid IGF-1 concentrations on day of egg collection were measured by RIA after acidification and extraction by reveresed phase chromatography. Patients receiving G.H. required fewer days and ampules of gonadotropins, developed more oocytes, and more embryos transferred. But, the differences were not statistically significant, except the duration of hMG treatment. Our data showed a significantly higher concentration of IGF-l in the serum, not in the follicular fluid, of patients treated with G.H. compared with control group. These data suggest that growth hormone treatment does not improve the ovarian response in women with limited ovarian reserve to gonadotropin stimulation for IVF.

• Journal of Embryo Transfer
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• v.14 no.2
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• pp.121-129
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• 1999

The objective of this study was to produce calves by transfer of embryos derived from slaughter house(SHD) and ultrasound-guided ovum pick-up (OPU). At 60 hrs after injection of 400 mg FSH dissolved in 25% polyvinylpyrrolidone(PVP) by single dose, ultrasound-guided follicular oocyte aspiration was ferformed. Day-7 and day-8 blastocysts produced by in vitro maturation (IVM), fertilization (IVF) and culture(IVC) of the oocytes derived from SHD and OPU were nonsurgically transferred into recipients. The results obtained were as follows. The cleavage rate and the development rate to blastocysts were not significantly (P<0.05) different between the oocytes obtained by SHD (72.9% vs. 34.1%) and OPU (75.9% vs. 38.4%). The oocyte recovery rate from the number of follicles by ultrasound-guided aspiration were not significantly (P<0.05) different between Holstein (61.7%) and Hanwoo (60.1%), but the rate of oocytes useful for IVF was significantly (P<0.05) higher in Hanwoo (69.3%) than Holstein (59.6%). The cleavage rate and the development rate to blastocysts was not significantly (P<0.05) different between Holstein (74.9% vs. 39.2%) and recipients on day 8 of estrus cycle resulted in 13 pregnancies (34.2%). One of them was sacrificed during gestation period due to mastitis and another was aborted spontaneous. The resulting 14 calves were morphologically normal at birth. Seventy eight fresh OPU-IVF embryos were transferred into 21 recipients on day 8 of estrus cycles, resulting in pregnancy of 12 recipients (41.4%). Two of them were sacrificed during gestation period due to mastitis and the other two were aborted. Nevertheless, the 11 OPU-calves have been born normally.

• Journal of Embryo Transfer
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• v.28 no.2
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• pp.157-162
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• 2013

Methoxychlor (MXC) was developed to be a replacement for the banned pesticide DDT. HPTE [2,2-bis (p-hydroxyphenyl)-1,1,1-trichloroethane], which is an in vivo metabolite of MXC, has strong oestrogenic and anti-androgenic effects. MXC and HPTE are thought to produce potentially adverse effects by acting through oestrogen and androgen receptors. Of the two, HPTE binds to sex-steroid receptors with greater affinity, and it inhibits testosterone biosynthesis in Leydig cells by inhibiting cholesterol side-chain cleavage enzyme activity and cholesterol utilisation. In a previous study, MXC was shown to induce Leydig cell apoptosis by decreasing testosterone concentrations. I focused on the effects of MXC on male mice that resulted from interactions with sex-steroid hormone receptors. Sex-steroid hormones affect other organs including the kidney and liver. Accordingly, I hypothesised that MXC can act through sex-steroid receptors to produce adverse effects on the testis, kidney and liver, and I designed our experiments to confirm the different effects of MXC exposure on the male reproductive system, kidney and liver. In these experiments, I used pre-pubescent ICR mice; the puberty period in ICR mice is from postnatal day (PND) 45 to PND60. I treated the experimental group with 0, 100, 200, 400 mg MXC/kg b.w. delivered by an intra-peritoneal injection with sesame oil used as vehicle for 4 weeks. At the end of the experiment, the mice were sacrificed under anaesthesia. The testes and accessory reproductive organs were collected, weighed and prepared for histological investigation. I performed a chemiluminescence immune assay to observe the serum levels of testosterone, LH and FSH. Blood biochemical determination was also performed to check for other effects. There were no significant differences in our histological observations or relative organ weights. Serum testosterone levels were decreased in a dose-dependent manner; a greater dose resulted in the production of less testosterone. Compared to the control group, testosterone concentrations differed in the 200 and 400 mg/kg dosage groups. In conclusion, I observed markedly negative effects of MXC exposure on testosterone concentrations in pre-pubescent male mice. From our biochemical determinations, I observed some changes that indicate renal and hepatic failure. Together, these data suggest that MXC produces adverse effects on the reproductive system, kidney and liver.

• Clinical and Experimental Reproductive Medicine
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• v.40 no.3
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• pp.131-134
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• 2013

Objective: To evaluate the effect of the addition of estradiol to luteal progesterone supplementation in GnRH antagonist cycles for infertile patients undergoing IVF/ICSI. Methods: One hundred and ten infertile patients, aged 28 to 39 years, were recruited for this prospective randomized study. They were randomly assigned to receive vaginal progesterone gel (Crinone) along with 4 mg estradiol valerate (group 1, n=55) or only Crinone (group 2, n=55) for luteal support. A GnRH antagonist multiple dose protocol using recombinant human FSH was used for controlled ovarian stimulation (COS) in all of the subjects. The COS results and pregnancy outcomes of the two groups were compared. Results: Group 1 and 2 were comparable with respect to the patient characteristics. The COS and IVF results were also comparable between the two groups. There were no differences in the clinical pregnancy rate (PR) and multiple PR between the two groups. However, the embryo implantation rate were significantly higher in group 1 than that in group 2 (22.2% vs. 13.3%, p=0.035). The incidence of luteal vaginal bleeding (LVB) was significantly lower in group 1 (7.4% vs. 27.8%, p=0.010). Conclusion: The addition of estradiol to luteal progesterone supplementation in GnRH antagonist cycles reduces the incidence of LVB and increases the embryo implantation rate in infertile patients undergoing IVF/ICSI.