• Clinical and Experimental Reproductive Medicine
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• v.26 no.2
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• pp.239-249
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• 1999

The role of amino acids in culture media for IVF-ET was examined in a total of 76 cycles. Patients received clomiphene citrate (CC) followed by hMG or GnRH-a combined with gonadotropins (FSH/hMG) for controlled ovarian hyperstimulation. Severe male (<$4{\times}10^6$ motile sperm) or age factor (>39 y) patients were excluded in this study. Pregnancy was classified as clinical if a gestational sac or fetal cardiac activity was seen on ultrasound. No significant differences were found in age, duration of infertility, follicle size, the level of $E_2$ on the day of hCG injection, the mean number of oocytes retrieved, total motile sperm count, fertilization rate and the mean number of embryos transferred between bHTF (without amino acids) and mHTF (with amino acids) groups. However, total ampules of gonadotropins were higher (p<0.01) in mHTF group than bHTF group. Significantly (p<0.05) more clinical pregnancies were recorded in mHTF group (13/30) compared with bHTF group (9/46). The multiple pregnancy rates were 11.1% in bHTF group and 7.7% in mHTF group. There were one ectopic pregnancy in mHTF group and one heterotopic pregnancy in bHTF group. Abortion rates were 22.2% in bHTF group and 7.7% in mHTF, respectively. The ongoing pregnancy or livebirth rate was significantly (p<0.05) higher in mHTF group (12/30) than bHTF group (7/46). These results suggest that the addition of amino acids in culture media is essential for culture of zygotes in vitro and adjustment of energy substrates in phosphate-free culture media appears to be beneficial for human IVF-ET procedure.

• 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.38 no.1
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• pp.37-41
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• 2011

Objective: To compare the effectiveness and convenience of a pen device for the self-administration of follitropin ${\beta}$ with a conventional syringe delivering follitropin ${\beta}$ solution in patients undergoing IVF-ET. Methods: GnRH agonist long protocol was used for controlled ovarian stimulation (COS) in all subjects. A total of 100 patients were randomized into the pen device group or the conventional syringe group on the first day of COS. Local tolerance reactions were assessed within 5 minutes, at 1 hour and at 3 hours after each injection. On the day of hCG injection, patients were asked to rate their overall pain and convenience experienced with self-injection on a visual anlaogue scale (VAS). Results: There were no differences in patients' characteristics between the two groups. The duration of COS was significantly shorter in the pen device group than in the conventional syringe group. Patients included in the pen device group needed a significantly smaller amount of follitropin ${\beta}$. However, no differences between the two groups were found in IVF results and pregnancy outcome. The incidence of local pain within 5 minutes, at 1 hour and at 3 hours after the injection was significantly lower in the pen device group. VAS scores indicated that injections using the pen device were significantly less painful and more convenient. Conclusion: The pen device for self-administration of follitropin ${\beta}$ is less painful, safer and more convenient for the patients, and can be more effective because of the shorter duration and smaller dose of follitropin ${\beta}$ when compared with the conventional syringe.

• Development and Reproduction
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• v.22 no.1
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• pp.85-94
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• 2018

Endocrine disruptors have been concerned in toxicology but now challenged as physiological point especially concerned with exposing dose and period. In this study the low-dose chronic administration of di(2-ethylhexyl) phthaltae (DEHP) during reproductive period was examined to evaluate the possible roles. Adult male and female CD-1 mice were exposed to DEHP with drinking water containing $133{\mu}g/L$ and $1,330{\mu}g/L$ DEHP in water according to OECD 433 guide line and sacrificed just after weaning. The weights of uterus and ovary were decreased by drinking of $1,330{\mu}g/L$ DEHP water. There was not adverse effects on either accumulated mating rate and mating rate depend on estrus stage, pregnancy duration, and sex ration at birth. However, the accumulated rate of successful delivery and litter size were significantly high at $1,330{\mu}g/L$ DEHP water. The number of epididymal sperm was significantly increased by drinking of $1,330{\mu}g/L$ DEHP water. In addition, the number of follicles (primary, secondary, tertiary) were more many than control at $1,330{\mu}g/L$ DEHP water drunk mother. Though further studies are needed to identify what are the mechanism of DEHP in folliculogenesis and spermatogenesis. From this study we firstly report the effect of low-dose chronic administration of DEHP with drinking could change the ovarian follicle population size and spermatogenesis rate. Put together, those finding is different from previous high-dose effects and suggest the physiological role of DEHP in gonads and uterus.

• Journal of Veterinary Clinics
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• v.15 no.1
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• pp.131-139
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• 1998

Progesterone을 함유하고 있는 CIDR(Controlled Internal Drug Release)의 질내삽 입은 황체기를 인위적으로 연장시킬 수 있다. CIDR의 삽입이,삽입시 존재했던 우세난포 (dominant follicle)의 반응과 난포의 발육반응 그리고 2회 또는 3회의 난포주기를 가지고 있는 처려우에서 CIDR의 삽입기간동안 난포의 성장 및 발육에 어떠한 영향을 미치는가를 비교검 토하기 위하여 배란후 16일째의 처녀우 4마리에 7일동안 CIDR를 삽입하였다. CIDR의 삽입 은 발정의 발현을 억제시켰으며 그리고 발정주기의 길이를 정상 발정주기보다 유의성있게 연 장시켰다($26.3{\pm} 0.5 vs 20.8{\pm}$ 1.5일, p<0.05). CIDR의 삽입시 혈장 progesterone 농도는 $3.6{\pm}$ 2.7 ng/ml 이었으며, 17일과 23일 사이에는 2.1-4.4 ng/ml($3.6{\pm}1.2 ng/ml$) 사이를 유지했다. 혈 장 estradiol-179의 농도는 난포의 발육 및 배란전 배란난포의 성숙을 나타내는 특징적인 변화 양상을 나타래었다. 4마리의 처녀우중 2마리는 CIDR 삽입전 발정주기당 2회의 난포주기를 가진 반면, 나머지 2마리는 주기당 3회의 난포주기를 가졌다. 그렇지만 CIDR의 삽입기간동안 모든 처녀우는 주기당 3회의 발정주기를 가졌다. CIDR의 삽입전 발정주기당 3회의 난포주기 를 갖는 처녀우에서 CRR의 삽입은 세 번째 난포주기에서 배란성 우세난포의 우세기 (dominant phase)를 연장시켰다. 3회의 난포주기를 갖는 2마리에서 CIDR의 삽입후 배란난포 는 존속시간과 우세기가 유의성있게 연장되었다. CIDR의 삽입전 발정주기당 2회의 난포주기 를 갖는 다른 2마리의 처녀우에서 CIDR의 삽입후 우세난포는 곧바로 퇴행되었고 새로운 난 포주기를 형성하였으며, 우세난포의 우세기와 배란난포의 존속기간을 연장시키지 않았다. CRR의 삽입은 CIDR의 삽입후 이어지는 발정주기동안 난포의 발육 및 성장에 영향을 미치 지 않았으며 발정주기의 길이, 난포주기, 혈장 progesterone 및 estradiol-179 농도에 영향을 미치지 않았다. 결과적으로 황체기 후반부에 CIDR의 삽입은 CIDR삽입전 발정주기동안 3회 의 난포주기를 갖는 처녀우에서 배란성 우세난포의 발육과 배란까지의 기간을 연장시켰고 2회 난포주기를 갖는 처녀우에서는 우세난포를 곧바로 퇴행시킨후, 새로운 난포주기를 형성 하였다.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.8
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• pp.1112-1121
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• 2019

Objective: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Methods: First, we developed an in vitro model to study the response of bovine cumulusoocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in $10{\mu}g/mL$ of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-${\kappa}B$); and the concentrations of interleukin (IL)-$1{\beta}$, tumor necrosis factor (TNF)-${\alpha}$, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Results: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-${\kappa}B$. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-$1{\beta}$, TNF-${\alpha}$, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. Conclusion: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.36 no.4
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• pp.265-274
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• 2009

Objective: MicroRNAs (miR) are known to repress target genes at post-transcriptional level and play important roles in development and maturation of cell. However, the expression profiles of miR during ovarian follicle maturation have not been fully elucidated. Here, we designed this study to investigate the expression profiles of miR in oocytes and granulose cells (G-cells) after in vitro culture according to gonadotropins and adding hCG. Methods: Ovaries from 12-day-old mice (C57BL6) were removed and preantral follicles were isolated and cultured in $20\;{\mu}L$-drop of culture media with supplementation of either rFSH, rLH, or rFSH+rLH. After their full maturation, follicles were incubated with rhCG and rEGF. RNA was isolated from oocytes and G-cells, and real-time PCR were performed with primers of miR known to be expressed in the mouse ovary (mmu-miR-16, -miR-27a, -miR-126, -miR-721). Results: FSH+LH group showed the highest ovulation and MII rates among gonadotropin groups. The profiles of miRs in oocytes and G-cells differed according to gonadotropin groups and adding hCG. The profiles of miRs showed divergent changes between oocytes and G-cells. Conclusion: miR expression profiles are altered by gonadotropins and supplementation of hCG during in vitro maturation of murine follicles. Target gene study must be necessary to validate these findings.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.119-124
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• 2001

This study was performed to investigate the effects of the peptide to carrier ratio on the immune and biological functions to inhibin immunization in Hanwoo. A peptide sequence kom the alpha -subunit (19~32 peptide) of porcine inhibin was synthesized for antigen and conjugated to human serum albumin(HSA) for carrier protein. Anti-inhibin sera(AI) were produced 52 day later from rabbit after injection of inhibin-$\alpha$ -subunit peptide conjugator for antigen with the interval of 2 weeks. Immune-blotting analysis using antibody specific fur inhibin-$\alpha$ subunits revealed that the inhibin was detected at 1.0 cm bovine follicular fluid(bFF). However, each stage of corpus lutea and 0.1 cm of follicular fluid were not detected. The maximal contents of estradiol-17 $\beta$ in Hanwoo ovarian follicular fluid were detected at 2.0 cm of follicular size(diameter), but the mean total contents of these hormone decreased significantly with decreasing diameter of follicles. However, progesterone contents of follicular fluid were high at 1.0 cm of follicle. Progesterone secretion by Hanwoo granulosa cell cultured for 48 hr in vitro was significantly (p＜0.05) inhibited in 5% bFF and 5% bFF + 5% AI addition group compared with control group. Estradiol-17 $\beta$ secretion by Hanwoo granulosa cell cultured for 48 hr in vitro was significantly (p＜0.05) increased in 5% AI and 5% AI + 5% bFF addtion group compared with control group. However, the groups added 5% AI were not changed compared to control groups in progesterone and estradiol-17 $\beta$. Taken together, we suggested that inhibin in the mature FF plays a pivotal role on the biosynthesis of steroid hormone of follicular cells during follicular development.

• Journal of Animal Science and Technology
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• v.46 no.4
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• pp.571-584
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• 2004

It is known widely that granulosa cell apoptosis leads follicular atresia and macrophages exert their effects directly and/or indirectly from the initiation to the completion of follicular atresia by phagocytosis of apoptotic bodies and secretion of various cytokines. However, the site of initiation, propagation routes and the elimination methods of apoptotic bodies, and the time and methods of penetration of macrophages into the follicles are not known completely. Using pig(Yorkshire-breed) ovary, immunohistochemical studies with TUNEL for apoptotic bodies and pig macrophage monoclonal antibody 4E9 for macrophages, and light and transmission electron microscopic observations were performed. In the pig, follicular atresia began with the granulosa cell apoptosis, and the apoptosis of theca intema cells occured at the same time. The apoptosis of granulosa cells initiated randomly within the granulosa cell layer and propagated rapidly into the whole layer. Ultrastructura1ly, apoptotic granulosa cells showed characteristic pyknotic and deformed nucleus and intracytoplasmic vesicles. Apoptotic bodies were eliminated by intact granulosa cells and macrophages. Intact granulosa cells ingested apoptotic bodies transiently, soon after they fell into the apoptosis. Finally, apoptotic bodies and degenerated oocyte were phagocytosed by macrophages. Macrophages entered the ovarian follicle at the time of initiation of granulosa cell apoptosis, and migrated with the progression of apoptosis. By elimination of theca cells, macrophages contributed the completion of follicular atresia These results will provide valuable informations on the study of the interrelation between macrophage and ovarian follicular atresia.

• Applied Microscopy
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• v.31 no.1
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• pp.19-35
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• 2001

Outlines for plasma $estradiol-17\beta$, components, electrophoretic patterns, and ultrastructural changes were obtained in female rainbow trout (Oncorhynchus mykiss) during the seasonal reproductive cycles. Plasma $estradiol-17\beta$ under the natural conditions, exhibited distinct seasonal variation, peaking very late in vitellogenic season during September, decreasing gradually the halt of spawning in December, and ultimately falling during the early stages of seasonal ovarian recrudescence in February and March. This change in $estradiol-17\beta$ appeared to stimulate vitellogenin production as evidenced by increases in plasma calcium, phosphorus, glucose, albumin and total protein levels. The electrophoretic patterns of late maturing or spawning oocytes were stained more intensively than those of late perinucleolus oocytes (molecular weights of approximately 70,000 and 200,000). Two protein bands were found in the SDS-PAGE separation, coincident with the $estradiol-17\beta$ hormone peak. Gonadosomatic indices (GSI) significantly increased from October to January, and showed the highest peak in January, coinciding with the numerically abrupt increase of ripe ova in female. A positive correlation (r=0.701, p<0.01) was established between plasma $estradiol-17\beta$ levels and the gonadosomatic index during the prespawning. The highest level of hepatosomatic index (HSI) observed in December. During the breeding season (December), the gonadotropes were large and filled with GTH-containing inclusions such as granules and globules. The vitellogenic phase began as late perinurleolus oocytes became transformed into early maturing oocytes through the accumulation of yolk, and oocytes reached the late maturing stages as the ooplasm was completely packed with yolk. Marked ultrastructural changed in the granulosa cells during nuclear migration involve the dilation of the rough endoplasmic reticulum and the appearance of the rod-shaped mitochondria with tubular cristae. Microvilli (finger-like projections), from the zona radiata and from the oocyte grew, and made contact with each other in the pore canals of the zona radials during vitellogenesis, but were withdrawn as the zona radiata became more compact and devoid of pore canals during oocyte maturation. The zona radiata grew to a tripartite structure such as an outer thin homogeneous layer, and two inner thick helicoidal layers (zona radials interna and zona radiata externa). Under the normal conditions, the ovarian follicle influenced the histological development and periodical secretion of the hormones , sufficient for a oogenesis and gonadal steroid production.