• Clinical and Experimental Reproductive Medicine
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• v.26 no.3
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• pp.389-398
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• 1999

Objective: This study was designed to evaluate the effects of endogenous LH surge, GnRH agonist (GnRH-a) or human chorionic gonadotropin (hCG) as ovulation trigger on pregnancy rate by intrauterine insemination (IUI). Method: Patients received daily 100 mg of clomiphene citrate (CC) for 5 days starting on the third day of the menstrual cycle followed by human menopausal gonadotropin (hMG) for ovulation induction. Follicles larger than >16 mm in diameter were present in the ovary, frequent LH tests in urine were introduced to detect an endogenous LH surge. Final follicular maturation and ovulation were induced by GnRH-a 0.1 mg (s.c.) or hCG $5,000{\sim}10,000$ IU (i.m.) administration except natural ovulation. Pregnancy was classified as clinical if a gestational sac or fetal cardiac activity was seen on ultrasound. Results: There were no differences in age, duration of infertility and follicle size, but more ampules of hMG were used in GnRH-a group compared to hCG 10,000 IU treated group (p<0.05). Lower level of estradiol ($E_2$) on the day of hCG or GnRH-a injection was observed in hCG 10,000 IU group than other treatment groups (p<0.01). The overall clinical pregnancy rate was 19.8% per cycle (32/162) and 22.2% per patient (32/144). Pregnancy rate was higher in natural-endogenous LH surge group (37.5%, 9/24) than GnRH-a (18.8%) or hCG treated group (20.9% & 13.9%), but this difference was not statistically significant. No patient developed ovarian hyperstimulation. Abortion rate was 22.2% (2/9) in hCG 5,000 IU group. Delivery or ongoing pregnancy rate was 37.5% (9/24), 18.8% (3/16), 16.3% (7/43) and 13.9% (11/79) in endogenous LH surge, GnRH-a, hCG 5,000 IU and hCG 10,000 IU treatment groups, respectively. Conclusion: These results support the concept that use of natural-endogenous LH surge in stimulated cycles may be more effective to obtain pregnancies by IUI than GnRH-a or hCG administration.

• Clinical and Experimental Reproductive Medicine
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• v.35 no.2
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• pp.111-118
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• 2008

Objective: The purpose of the present study was to examine the hormonal regulation of RGS-2 in the rat ovary. Methods: Immature rats were injected with 10 IU of PMSG to induce multiple growth of preovulatory follicles and 10 IU of hCG to induce ovulation. Northern blot analysis performed for gene expression and in situ hybridization performed for mRNA localization. Results: Northern blot analysis revealed that pregnant mare's serum gonadotropin (PMSG) treatment did not affect RGS-2 mRNA levels. In contrast, human chorionic gonadotropin (hCG) treatment of PMSG-primed rats resulted in an increase in RGS-2 expression within $1{\sim}3\;h$. The major cell-types expressing RGS-2 mRNA were oocytes regardless of follicle size. Interestingly, hCG treatment caused the stimulation of RGS-2 gene expression in granulosa cells of preovulatory and growing follicles. In contrast, cell types expressing RGS-2 protein were theca cells regardless of hCG treatment. Like in vivo, treatment of preovulatory granulosa cells with LH in vitro stimulated RGS-2 levels within 1 h. Interestingly, GnRH antagonist II enhanced the stimulatory action of LH. Conclusion: The present study demonstrates the LH/hCG induction of RGS-2 in preovulatory granulosa cells and suggests a role of RGS-2 in Gq protein signaling pathway during ovulation.

• Journal of Veterinary Clinics
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• v.21 no.2
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• pp.115-121
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• 2004

The purpose of this study was to ascertain the breeding efficiency in Thoroughbred mare. A total of 106 mares were investigated for the status of follicle (462 cases), ovulation (179 cases) and pregnancy (346 cases). Of total examination, 46.8% was follicle measure to determine breeding time, and mating rate per cases examined was 39.9%. There was no correlation between reproductive results and size of follicles or endometrial edema or degrees of teasing alone. 143 cases were ovulated among 179 cases which were performed ovulation examination, and ovulation rate and fertilization rate per mating times were 79.9% and 39.0%, respectively. The use of hCG(human chorionic gonadotropin), to facilitate ovulation, presented to increase occurrence of double ovulations and twin fertilizations In conclusion, though more examination to estimate the optimal breeding time and higher mating rate was performed, fertilization rate per mating times was lower and then reproductive efficiency also became decreased. Therefore, it seemed that accurate examination of reproductive tracks, appropriate teasing programme and hCG administration before ovulation were of help to improve ovulation rate and fertilization rate.

• Clinical and Experimental Reproductive Medicine
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• v.18 no.2
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• pp.153-162
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• 1991

The present study was undertaken to clarify the role of calcium ion as a factor for the maturation of follicle-enclosed mouse oocytes. Follicles were isolated with two sharp needles under a stereomicroscope from mouse(ICR) ovaries which were treated PMSG 5 IU 45 hours previously. Isolated follicles were cultured for 14-16 hours in an organ culture system at $37^{\circ}C$, 5% $CO_2$ in air and in a 100% humidified incubator by treatment of hCG, EDTA and $^{45}Ca^{++}$. Culture medium was Modified Hank's Balanced Salt Sol. (MHBS) and addition of hCG (human chorionic gonadotropin) was made into two doses level 0.4 IU and 0.8IU from the stock sol. and also $^{45}Ca^{++}$ was treated in the culture medium. To explain the role of calcium, calcium chelating agent EDTA was treated to the culture of the mouse follicle-enclosed oocytes. Two observations were made in the present study; nucleus phase and $^{45}Ca^{++}$ uptake into the oocyte. HCG induced oocyte maturation in the follicle about two folds as much as the control group, whereas there is no difference in oocyte maturation between 0.4 IU and 0.8 IU of hCG. Optimum level of hCG seems to be 0.4 IU/ml in the mouse follicle culture. HCG stimulated $^{45}Ca^{++}$ uptake into the oocyte of the follicles by two folds. $^{45}Ca^{++}$ uptake in the control group is about 2.5 folds in comparison of the EDTA(1.71mM) treated group. However, calcium uptake in the EDTA treated groups tends to increase depending on the decrease of EDTA concentration. These observations suggest that firstly, hCG stimulates maturation of the oocyte of the follicle, secondly, $Ca^{++}$ influx is induced by hCG and thirdly, $Ca^{++}$ influx by the treatment of EDTA decreases as a dosage-dependent process. This $Ca^{++}$ uptake may take place by the changes of permeability which was induced by hCG treatment. That is, $Ca^{++}$ influx may trigger the resumption of oocyte maturation. It is further necessary in the future study how this $Ca^{++}$ uptake is induced by hCG and increases permeability of the follicle and oocyte.

• Journal of Life Science
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• v.24 no.10
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• pp.1039-1045
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• 2014

This study analyzed whether human chorionic gonadotropin (hCG) induces ER stress via the IRE/XBP1 pathway in mouse Leydig tumor (mLTC-1) cells. In a previous study, we demonstrated that the unfolding protein response (UPR) plays an important role in the expression of steroidogenic enzymes by modulating the ATF6 pathway, as well as ER stress-mediated apoptosis in hCG-stimulated Leydig cells. Although UPR signaling has been reported to regulate the IRE1/XBP1 pathway, it is not known whether hCG-induced ER stress in Leydig cells can activate the pathway. To investigate the activation of the IRE1/XBP1 pathway in mLTC-1 cells after hCG treatment, we performed a Western blot analysis to detect the phospho-IRE1 protein and an RT-PCR analysis to validate splicing of XBP1 mRNA. We used ER stress-activated indicator (ERAI) constructs for monitoring the activity of IRE1 and then analyzed by fluorescence microscopy and flow cytometry. The expression levels of the phospho-IRE1 protein markedly increased in response to the hCG treatment. In the mLTC-1 cells transfected with an F-XBP1-venus/F-$XBP1{\Delta}DBD$-venus construct, the hCG treatment led to the appearance of green fluorescent cells and detectable fluorescence in the nucleus and cytosol, respectively. In addition, splicing of XBP1 mRNA significantly increased after the hCG treatment. Taken together, these results indicate that hCG-induced ER stress leads to activation of the IRE1/XBP pathway in Leydig cells.

• Laboraroty Animal Research
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• v.34 no.4
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• pp.211-215
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• 2018

The following experiments were designed to examine the effect of serum of spayed dogs on superovulation response in mice and rats. In Experiment 1, female mice at diestrus (n=30) were divided into three equal groups and superovulated with either administration of 5 IU pregnant mare serum gonadotropin (PMSG) or recombinant follicle stimulating hormone (rFSH) (reducing dose from 2.5 to 0.5 IU) and 5 IU human chorionic gonadotropin (hCG) administered 48h later. Serum of spayed dogs was administered intraperitoneally at a reduced dose from 0.1 to 0.025 mL in a 48 h period. In Experiment 2, female rats (n=30) at diestrus stage were divided into three equal groups. Superovulation was induced using either 30 IU PMSG, or a dose reduced from 5 to 1 IU rFSH and 25 IU hCG administered 48h later. Serum of spayed dogs was administered in a reduced dose from 0.6 to 0.1 mL in a 48 hour period. Female mice and rats were mated 24 h following hCG administration. On day 14 after mating, animals were euthanized and ovarian sections were fixed for histopathological evaluation and corpus luteum (CL) counting. No significant difference observed in mean (${\pm}SEM$) number of CLs between the PMSG group and the mice that received serum of spayed dog ($10.4{\pm}1.3$ vs $9.2{\pm}1.0$). Mean (${\pm}SEM$) number of CLs tended to be lower in rats that received serum of spayed dog than those of rats which received either PMSG or rFSH ($15.1{\pm}1.9$ vs $23.6{\pm}3.1$ and $23.1{\pm}2.9$, P=0.06, respectively). In conclusion, serum of spayed dogs is able to induce a superovulatory response in mice and rats.

• Development and Reproduction
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• v.27 no.3
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• pp.149-157
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• 2023

We investigated the involvement of autophagy with steroidogenesis in testicular Leydig cells. Human chorionic gonadotropin (hCG)-stimulated T production in Leydig cells was not remarkably altered in the presence of an autophagy inhibitor 3-methyladenine (3-MA). Although pretreatment with 3-MA demonstrated a tendency to decrease hCG-induced T production, the differences were significant only at a higher time point of 24 h following hCG. Microtubule associated protein light chain 3 (LC3)-II was detectable in the control cells in all the experiments. The hCG-induced increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleave (P450scc) protein levels were not significantly altered by 3-MA. Leydig cells isolated from immature rat testes 12 h following hCG treatment showed relatively increased levels of LC3-II protein compared to the control group. Furthermore, LC3-II levels shown in these cells reached almost the identical to those from normal adult testes. However, LC3-II protein levels were almost comparable or even slightly lower than the controls at 48 h following hCG. Expression of StAR and P450scc was upregulated at both 12 and 48 h after hCG. We also used MA-10 cells, the mouse Leydig cell line, in this experiment. When dibutyryl cyclic-AMP was treated with MA-10 cells, P4 levels were significantly increased in the cell culture medium. However, P4 levels tended to decrease in the presence of 3-MA, but the difference was not statistically significant. This was consistent with the results of the rat Leydig cell experiments. Together, we believe that although autophagy participates in steroidogenesis and enhances steroidogenic efficacy of Leydig cells, it may not be a decisive cellular process for steroidogenesis, specifically in the mature Leydig cells.

• Clinical and Experimental Reproductive Medicine
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• v.19 no.1
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• pp.41-48
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• 1992

Serum level of ${\beta}$ subunit of human chorionic gonadotropin (${\beta}-hCG$) was studied to evaluate its predictability of pregnancy outcome in 98 in vitro fertilization and embryo transfer(IVF-ET) patients using gonadotropin-releasing hormone(GnRH) agonist. Serial serum ${\beta}-hCG$ levels were established for 42 singleton pregnancies, 20 normal multiple pregnancies, 18 preclinical abortions, 14 clinical abortions and 4 ectopic pregnancies. In comparison to normal singleton pregnancies, multiple pregnancies showed significantly higher ${\beta}-hCG$ levels on the post-ET day 10 to 13 and day 24 to 25. Clinical abortions did not show significantly lower ${\beta}-hCG$ levels in early pregnancy except the post-ET day 16-17, but showed significantly lower ${\beta}-hCG$ levels from the post-ET day 22, compared with singleton pregnancies. Preclinical abortions showed significantly lower ${\beta}-hCG$ levels than those of singleton pregnancies. Ectopic pregnancies showed lower ${\beta}-hCG$ levels than those of singleton pregnancies without statistical significance. In conclusion, determination of serum ${\beta}-hCG$ level in early pregnancy is a useful tool for the prediction of preclinical abortions and multiple pregnancies and serial measurement of serum ${\beta}-hCG$ levels will be helpful in predicting clinical abortion.

• Clinical and Experimental Reproductive Medicine
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• v.25 no.1
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• pp.9-16
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• 1998

This study was performed to investigate the stimulating effect on oocyte maturation and cumulus cell expansion in TC199 media by human cord serum (HCS) supplementation. Immature mouse oocyte cumulus complexes (OCCs) were cultured in TC199 media supplemented with bovine serum albumin (BSA), HCS and human chorionic gonadotropin (hCG) instead of luteinizing hormone (LH) respectively, and the expression of cumulus expansion and oocyte maturation were observed. After 4hr and 24hr culture with or without OCCs, media containing 0.4% BSA, 10% HCS and 10 IV hCG respectively were collected and analyzed for changing concentrations of estradiol $(E_2)$, progesterone $(P_4)$, testosterone (T), and $PGF_{2\alpha}$. There were no elevation of $E_2$, T, and $PGF_{2\alpha}$ by OCCs culture, but minute elevation of $P_4$ level by 24hr OCCs culture in hCG supplementation (p=0.048). The stimulating pattern of cumulus expansion of OCCs by HCS and hCG supplementation was similar to our previously report using Ham's F-10 media, however oocyte maturation rates after 24hr OCCs culture in all media were increased by $20\sim30%$ compared to Ham's F-10 media. These results suggest that LH in HCS induce cumulus expansion probably by $P_4$ secretion of OCCs, and TC199 is efficient media for immature mouse oocyte maturation.

• Clinical and Experimental Reproductive Medicine
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• v.21 no.2
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• pp.165-176
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• 1994

This study was carried out to investigate the effect of the timing and dose of human chorionic gonadotropin(hCG) on oocyte recovery, in vitro fertilization, and preimplantation development in superovulation of mouse. F1 hybrid($C57BL{\times}CBA$) mice were obtained and superovulation was induced in female mice by sequential intraperitoneal injection of PMSG and hCG. In the first series of experiments, mice received 5 IU of PMSG given intraperitoneally, and 48 hours later were injected 1 IU, 5 IU, or 10 IU of hCG respectively. In the second series of experiments, mice received 5 IU of PMSG given intraperitoneally and were injected 5IU of hCG 36, 48, or 60 hours later respectively. 1. When the mice received 5 IU of PMSG given intraperitoneally and 48 hours later were injected 1 ItT, 5 IU, or 10 IU of hCG respectively, there were no differences in the total number of the oocytes obtained from the three experimental groups. When the cultures were examined 48 hrs after the termination of insemination the proportion of unfragmented oocytes which had developed over two-cell stage was observer to be lowest in 10 IU hCG group. When the cultrues were examined 120 hour after termination of insemination the proportion of embryos which had developed to the blastocyst stage was observed to be significantly higher in 10IU hCG group than 5IU hCG group(p<0.05), but there was no difference between 10 IU hCG group and 1IU hCG group. 2. When the mice received 5 IU of PMSG and were injected 5 IU of hCG 36, 48, or 60 hours later respectively, there were no differences in the total number of oocytes obtained from the three experimental groups. When cultures were examined 48 hour after the termination of insemination the proportion of unfragmented oocytes which had developed over two-cell stage was observed to be significantly lower in 36 hour interval group than 48 hour interval and 60 hour interval group(p<0.05). When the cultures were examined 120 hour after termination of insemination the proportion of embryos which had developed to the blastocyst stage was found to be higher in 60 hour interval group than 36 interval or 48 hour interval group (P<0.05), and the proportion of hatching blastocyst was found to be higher in 60 hour interval group as well. In this study, it was concluded that the administration of adequate dose of hCG, and long (60 hour) PMSG-hCG interval were necessary in superovulation of mice($C57BL{\times}CBA$) in order to get a large number of oocytes which had an early oocytes which had an early embryonic developmental capability when fertilized in vitro, and especially it had better have been avoided to administer a large dose of hCG.