• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.487-499
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• 2016

The aim of the present study was to examine the effects of testosterone (T) and estradiol-$17{\beta}$ ($E_2$) on the production of progesterone ($P_4$) by granulosa cells, and of the $E_2$ on the production of $P_4$ and T by theca internal cells. In the first experiment, granulosa cells isolated from the largest ($F_1$) and third largest ($F_3$) preovulatory follicle were incubated for 4 h in short-term culture system, $P_4$ production by granulosa cells of both $F_1$ and $F_3$ was increased in a dose-dependent manner by ovine luteinizing hormone (oLH), but not T or $E_2$. In the second experiment, $F_1$ and $F_3$ granulosa cells cultured for 48 h in the developed monolayer culture system were recultured for an additional 48 h with increasing doses of various physiological active substances existing in the ovary, including T and $E_2$. Basal $P_4$ production for 48 h during 48 to 96 h of the cultured was about nine fold greater by $F_1$ granulosa cells than by $F_3$ granulosa cells. In substances examined oLH, chicken vasoactive intestinal polypeptide (cVIP) and T, but not $E_2$, stimulated in a dose-dependent manner $P_4$ production in both $F_1$ and $F_3$ granulosa cells. In addition, when the time course of $P_4$ production by $F_1$ granulosa cells in response to oLH, cVIP, T and $E_2$ was examined for 48 h during 48 to 96 h of culture, although $E_2$ had no effect on $P_4$ production by granulosa cells of $F_1$ during the period from 48 to 96 h of culture, $P_4$ production with oLH was found to be increased at 4 h of the culture, with a maximal 9.14 fold level at 6 h. By contrast, $P_4$ production with cVIP and T increased significantly (p<0.05) from 8 and 12 h of the culture, respectively, with maximal 6.50 fold response at 12 h and 6, 48 fold responses at 36 h. Furthermore, when $F_1$ granulosa cells were precultured with $E_2$ for various times before 4 h culture with oLH at 96 h of culture, the increase in $P_4$ production in response to oLH with a dose-related manner was only found at a pretreatment time of more than 12 h. In the third experiment, theca internal cells of $F_1$, $F_2$ and the largest third to fifth preovulatory follicles ($F_{3-5}$) were incubated for 4 h in short-term culture system with increasing doses of $E_2$. The production of $P_4$ and T by theca internal cells were increased with the addition of $E_2$ of $10^{-6}M$. These increases were greater in smaller follicles. These results indicate that, in granulosa cells of the hen, T may have a direct stimulatory action in the long term on $P_4$ production, and on $E_2$ in long-term action which may enhance the sensitivity to LH for $P_4$ production, and thus, in theca internal cells, $E_2$ in short term action may stimulate the production of $P_4$ and T.

• Asian-Australasian Journal of Animal Sciences
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• 제33권4호
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• pp.547-555
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• 2020

Objective: Apoptosis of ovarian granulosa cells (GCs) affects mammalian follicular development and fecundity. This study aimed to explore the regulatory relationship between microRNA-26a (miR-26a) and the 3β-hydroxysteroid-Δ24-reductase gene (DHCR24) gene in porcine follicular granular cells (pGCs), and to provide empirical data for the development of methods to improve the reproductive capacity of pigs. Methods: The pGCs were transfected with miR-26a mimic, miR-26a inhibitor and DHCR24-siRNA in vitro. The cell apoptosis rate of pGCs was detected by the flow cytometry. The secretion levels of estradiol (E2) and progesterone (P) in pGCs were detected by enzyme-linked immunosorbent assay. Double luciferase validation system was used to detect the binding sites between miR-26a and DHCR24 3'-UTR region. Qualitative real-time polymerase chain reaction and Western blotting were used to verify the DHCR24 mRNA and protein expression in pGCs, respectively, after transfecting with miR-26a mimic and miR-26a inhibitor. Results: Results showed that enhancement of miR-26a promoted apoptosis, and inhibited E2 and P secretion in pGCs. Meanwhile, inhibition of DHCR24 also upregulated the Caspase-3 expression, reduced the BCL-2 expression, promoted pGCs apoptosis, and inhibited E2 and P secretion in pGCs. There were the binding sites of miR-26a located within DHCR24 3'-UTR. Up-regulation of miR-26a inhibited DHCR24 mRNA and protein expression in pGCs. Conclusion: This study demonstrates that miR-26a can promote cell apoptosis and inhibit E2 and P secretion by inhibiting the expression of DHCR24 in pGCs.

• Clinical and Experimental Reproductive Medicine
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• 제15권2호
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• pp.135-147
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• 1988

To compare the stimulation effect of the ratio in follicle stimulating hormone and luteinizing hormone in induction of multiple follicular growth, the serum $E_2$ level, the diameter of follicle, number of aspirated follicles and cleavage rate of in vitro fertilized preovulatory oocytes as well as the pregnancy rate were evaluated. Forty one patients with irreparable tubal disease were stimulated by hMG(n=24) or FSH/hMG(n=17) for the purpose of in vitro fertilization and embryo transfer. The following results were obtained. 1. Serum estradiol($E_2$) levels on the day of hCG administration were $921.0{\pm}353.3\;pg/ml$ in hMG group and $1272.9{\pm}1060.6\;pg/ml$ in FSH/hMG group. The serum $E_2$ value of hMG group was significantly lower than that of FSH/hMG group. 2. The diameter of leading follicle by ultrasonogram on the day of hCG administration were $16.2{\pm}2.0\;mm$ in hMG group and $16.2{\pm}2.6\;mm$ in FSH/hMG group. No significant difference of follicle diameter between two groups was demonstrated. 3. The number of follicles with diameter above 10 mm by sonogram on the day of hCG injection were $3.91{\pm}2.32$ in hMG group and $6.52{\pm}3.86$ in FSH/hMG group. There was significant difference of number of follicles between two groups, (p< 0.01). 4. The number of oocytes found per patient at aspiration were $2.59{\pm}1.00$ in hMG group and 3. $76{\pm}2.31$ in FSH/hMG group. There was significant difference of number of aspirated oocytes between two groups. (p< 0.05). 5. The detection rate of preovulatory oocyte at aspiration were 68.4%(39/57) in hMG group (n=22) and 77.6%(38/49) in FSH/hMG group (n=13). 6. The cleavage rate of preovulatory oocyte at 44 hours after insemination were 74.4%(29/39) in hMG group(n=22) and 81.6%(31/38) in FSH/hMG group (n=13). When only hMG was used, one pregnancy was established in 15 patients to whom 29 zygotes were transferred. And a full term normal female baby was delivered by elective cesarean section. In the FSH/hMG group, five pregnancies out of 9 transferred patients were confirmed by serum ${\beta}-hCG$. Two pregnancies were spontaneously aborted before the 6th week of pregnancy. One patient aborted her baby at the 18th week of pregnancy because of incompetent internal os of the cervix. Two patients delivered two full term babies by elective cesarean section. From the above findings, paralell with the increase in the ratio of exogenous follicle stimulating hormone to luteinizing hormone, an increase in oocyte recovery was observed as well as an improvements in pregnancy rate. It was concluded that FSH enrichment early in the follicular phase had a beneficial effect in the controlled ovarian hyperstimulation.

• 한국발생생물학회지:발생과생식
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• 제1권2호
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• pp.189-202
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• 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.

• 한국수산과학회지
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• 제37권3호
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• pp.192-196
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• 2004

The in vitro effect of bisphenol A (BPA) on ovarian steroidogenesis of the longchin goby (Chasmichthys dolichognathus) was investigated. Oocytes taken during the maturing phase (vitellogenic, fully vitellogenic or germinal vesicle breakdown stage) were incubated with BPA (100 ng/mL) in the presence of exogenous precursor $^{3}H-17\alpha\;hydroxyprogesterone\;(^{3}H-17\alphaOHP).$ Steroids were extracted from the media and the isolated oocytes, and the extracts were separated and identified by thin layer chromatography and gas chromatography-mass spectrometry. The identities of the major metabolites were progestogens $[17{\alpha}-hydroxy,20{\alpha}-dihydroprogesterone\;(17{\alpha}20{\alpha}OHP)\;and\;17{\alpha}-hydrxy,20{\beta}-dihydroprogesterone\;(17{\alpha}20{\beta}OHP),$ androgens [androstenedione (A4) and testosterone (T)] and estrogens [estrone $(E_1)\;and\;estradiol-17{\beta}(E_2)].$ BPA treatment inhibited production of estrogens in all the maturing phases and progestogens in the germinal vesicle migrating stage. Percentage yield of estrogens was decreased with increased yield of androgens. In conclusion, BPA had an inhibitory effect on the conversion of $^3H-17\alphaOHP$ to estrogens and progestogens. These results demonstrate that BPA can act either estrogenic or anti-estrogenic effects.

• Nutrition Research and Practice
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• 제9권5호
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• pp.459-465
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• 2015

BACKGROUND/OBJECFTIVES: The effect of St. John's Wort extract (SJW) on MG-63 cell proliferation and trabecular bone loss induced by ovariectomy was examined. MATERIALS/METHODS: Proliferation, expression of estrogen receptor (ER) ${\alpha}$ and ER ${\beta}$, and gene expressions of osteoprotegerin (OPG), osteocalcin (OC) and alkaline phosphatase (ALP) were examined in MG-63 cells treated with or without SJW. Ovariectomized rats were treated with SJW at the dose of 100 or 200 mg/kg/day, ${\beta}$-estradiol-3-benzoate (E2), or vehicle only (OVX-C), and sham operated rats were treated with vehicle only (Sham-C). Serum ALP and C-telopeptide (CTX), and femoral trabecular bone loss were examined. RESULTS: SJW increased MG-63 cell proliferation and expression of ER ${\alpha}$ and ER ${\beta}$, and positive effect was shown on gene expressions of ALP, OC and OPG. SJW also showed estrogen like effect on bone associated with slowing down in trabecular bone loss. Histopathology by H&E showed rats treated with SJW displayed denser structure in metaphyseal region of distal femur compared with rats in OVX-C. SJW was shown to reduce serum CTX in OVX rats. CONCLUSION: The present study provides new insight in preventing estrogen deficiency induced bone loss of SJW and possibility for its application in bone health supplement.

• Clinical and Experimental Reproductive Medicine
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• 제25권2호
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• pp.217-231
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• 1998

The effectiveness of intrauterine insemination (IUI) combined with controlled ovanan hyperstimulation (COH) in the treatment of infertility with various etiologies was compared in a total of 152 cycles. Patients received a maximum of three IUI cycles for the treatment. Severe male ($<2\times10^6$ motile sperm) or age factor (> 39 y) patients were excluded in this study. Pregnancy was classified as clinical if a gestational sac was seen on ultrasound. The overall clinical pregnancy rate was 7.9% per cycle (12/152) and 9.7% per patient (12/124). The pregnancy rates were 0% in unstimulated natural (0/18), 7.5% in CC (3/40), 8.2% in CC+hMG (4/49), 5.9% in GnRH-a ultrashort (1/17), 5.9% in GnRH-a long (1/17) and 27.3% in dual suppression cycles (3/11), respectively. The pregnancy rate was higher in dual suppression cycle than other stimulated cycles, but this was not significant. The multiple pregnancy rates were 25.0% (2 twins and 1 triplet). No patient developed ovarian hyperstimulation. Abortion rates were 66.7% in CC (2/3) and 100% in ultrashort cycles (1/1). The livebirth rate was 5.9% per cycle (9/152) and 7.3% per patient (9/124). There were no differences in age, duration of infertility, follicle size, total ampules of gonadotropins and days of stimulation between pregnant and non-pregnant groups. However, significant(P<0.05) differences were observed in the level of estradiol $(E_2)$ on the day of hCG injection ($3,266.6{\pm}214.2$ vs $2,202.7{\pm}139.4$ pg/ml) and total motile sperm count ($212.1{\pm}63.4$ vs $105.1{\pm}9.9\times10^6$) between pregnant group and non-pregnant group. These results suggest that IUI combined with successful ovarian stimulation tends to improve the chance of pregnancy as compared to IUI without COH and a total motile sperm count may be considered predictive of the success for pregnancy.

• Asian-Australasian Journal of Animal Sciences
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• 제15권10호
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• pp.1403-1411
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• 2002

The aims of this study were, firstly, to analyze the biochemical compositions of serum and follicular fluid (FF) from prepubertal gilts after PMSG (1,000 IU) treatment. The concentrations of total proteins, lipids, cholesterol, glucose and sex hormones (progesterone, $P_4$; estradiol-$17{\beta}$, $E_2$; testosterone, T) were measured. Secondary, the effects of porcine FF (pFF) addition (40% and 100%) in IVM media and different culture conditions [Exp. 1: mBMOC-2+20% porcine serum (PS), fresh IVM medium, filtered IVMconditioned medium, or rabbit oviducts; Exp. 2: mBMOC-2+20%PS or stepwise medium replacement procedures (SMRP) cocultured with or without cumulus cells] on the in vitro development (IVD) of porcine oocytes were also examined. Results showed that no significant differences were found in total protein levels between serum and pFF from different sizes (large, >7 mm; medium, ~5-7 mm; small, <3-5 mm) of follicles (75-85 and 49-90 mg/dl; p>0.05). Total lipid concentrations remained constant in serum (395-472 mg/dl), and reduced significantly in the pFF from large follicles (287 mg/dl) at 132 h after PMSG treatment when compared to those at other time points (441-480 mg/dl). Basal cholesterol levels in serum and pFF at 12 h were similar (153-161 mg/dl), but increased at 36 h (186-197 mg/dl). Basal P4 and E2 levels in serum (0.1 ng/ml and 5.5 pg/ml) were low, but increased from 0.34 ng/ml and 12.13 pg/ml at 24 h to 0.81 ng/ml and 61.70 pg/ml at 98 h, respectively, after PMSG treatment (p<0.05). P4 levels increased linearly in pFF from large follicles during 12 through 132 h (138-1,288 ng/ml). A similar increase was also observed in $E_2$ levels (22-730 pg/ml) before 60 h post PMSG treatment, and then dropped afterwards (730-121 pg/ml). The development of the oocytes fertilized in 40% pFF-medium was greater than that in 100% pFF-medium group without gonaodtropin addition (31% vs 10%, p<0.05). However, both were lower than those in mBMOC-2+20%PS and in rabbit oviducts (p<0.05). When cocultured with cumulus cell monolayers, a greater cleavage rate was observed in the group cultured in filtered IVM-conditioned medium than the SMRP group (36% vs 18%, p<0.05). A similar phenomenon was also observed in the culture without cumulus cell monolayers (33% vs 19%, p<0.05). It is concluded that neither the fresh IVM nor filtered IVM-conditioned medium has positive effect on the IVD of oocytes. Coculture with cumulus cell monolayers and the SMRP were not beneficial to the development of IVF pig oocytes.

• Clinical and Experimental Reproductive Medicine
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• 제28권4호
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• pp.265-270
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• 2001

Objective : The aim of this study was to evaluate the outcome the GnRH antagonist (Cetrotide) short protocol in controlled ovarian hyperstimulation comparing with GnRH agonist long protocol. Materials and Method: From July 2000 to November 2001, 26 patients, 28 cycles were performed in controlled ovarian hyperstimulation by GnRH antagonist and GnRH agonist. GnRH antagonist (Cetrotide) was administered in 12 patients (14 cycles, Group 1) and GnRH agonist (Lucrin, Sub Q, Group 2) in 14 patients (14 cycles). Ovulation induction was performed by hMG (Pergonal) in group 1, and by Combo (Metrodine HP + Pergonal) in group 2. We compared the fertilization rate, good quality embryo, and clinical pregnancy rate between the two groups. Student-t test and Chi-square were used to determine statistical significance. Statistical significance was defined as p<0.05. Results: Ovarian hyperstimulation syndrome did not occurred in which estradiol (E2) level was $3874{\pm}809\;pg/ml$ and the number of retrieved oocytes was $18.4{\pm}2.4$. The number of used gonadotropin ampules was significantly decreased in Group 1 (26.0 vs. 33.1, p<0.04). There were no significant difference in the number of preovulatory oocyte ($10.6{\pm}6.9$ vs. $10.0{\pm}6.1$), fertilization rate ($74.8{\pm}23.4$ vs. $72.2{\pm}21.8$), good quality embryo ($58.7{\pm}23.6$ vs. $38.7{\pm}36.6$), and embryo transfer ($4.3{\pm}1.6$ vs. $4.4{\pm}1.6$). Although the age of the group 1 was older than the group 2 (34.4 vs. 30.8), there was no significant difference in clinical pregnancy rate (50.0% vs. 57.1%). Conclusions: We suggest that GnRH antagonist was a safe, effective, and alternative method in the controlled ovarian hyperstimulation, especially in PCOD patients who will be develop the ovarian hyperstimulation syndrome.

• 한국수정란이식학회지
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• 제29권1호
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• pp.35-41
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• 2014

Laparoscopic ovum pick-up (LOPU) is a convenient method for collecting oocytes in small ruminants. LOPU has the advantage of being a less invasive means of oocyte collection, thereby allowing for a repeated usage of the oocyte donor animals. A total of 25 Korean black goats were used in the winter season (December to February) and LOPU was applied to the goats which had been treated for superovulation more than two times during the last twelve months. Estrus was synchronized with an intravaginal insert containing 0.3 g progesterone for 10 to 12 days. Ovaries were hyperstimulated with eCG 1,000 IU oneshot, FSH with eCG (50 mg / 1,000 IU; 70 mg / 500 IU; 70 mg / 1,000 IU) oneshot or FSH multiple-shot with eCG oneshot ($20mg{\times}6/300IU$) given intramuscularly 72 h prior to LOPU. For these groups, the number of follicles (mean ${\pm}$ SEM) observed which developed to larger than 2 mm in diameter were $1.6{\pm}2.5$, $4.3{\pm}3.1$, $5.5{\pm}4.2$, $6.6{\pm}2.1$ and $8.8{\pm}7.8$, respectively. Oocytes were aspirated by using OPU needles and a vacuum pump. The overall oocyte retrieval rates were 41.4%. Oocytes were matured in TCM-199 supplemented with 10% (w/v) bovine serum albumin + $10{\mu}g/ml$ FSH + $1{\mu}g/ml$ $17{\beta}$-estradiol for 27 h at $39^{\circ}C$ in 5% $CO_2$ in air. Oocytes were parthenogenetically activated by ionomycin combined with 6-diethylaminopurine (6-DMAP). Total oocyte maturation and cleavage rate were 67.3% and 78.8%, respectively. In summary, LOPU is a useful oocyte collection method in Korean black goats that can provide immature oocytes for transgenesis or nuclear transfer.