• Reproductive and Developmental Biology
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• 제29권3호
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• pp.187-191
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• 2005

본 연구는 한우 난포란이 체외성숙된 환경의 변화를 단백질 측면으로부터 검토하기 위하여 체외성숙 배지와 세포질내 단백질 변화와 종류를 검토하였다. 그 결과 배지 내의 단백질 발현량은 배양 4.5시간째까지 감소하였고, 배양 13.5시간째까지는 변화가 없었다. 그러나 배양 $13.5\~18$시간 사이에 증가한 후 배양 18시간 이후에 다시 감소하는 경향이었다. 세포질내의 단백질 발현량은 배양 4.5시간째까지 증가하였고 배양 9시간째까지 급격히 감소하였다. 배양 9시간째부터 18시간째 까지는 단백질 발현량이 유사한 경향이었으나 배양 18시간째부터 24시간째까지 다시 증가하였다. 한편 체외성숙한 배지와 세포질을 2차원 전기영동하여 각각 298개 및 35개의 단백질 spot을 확인하였고, 그 중 배지에서는 28개, 세포질에서는 5개의 spot이 유의적인 변화를 확인하였다. 이들 spot 대한MALDI-TOP분석으로 배지와 세포질에서 각각 8개 및 1개의 단백질을 동정하였다. 그 종류는 aldose reductase, alpha enolase, apolipoprotein A-1 precursor, 43M1a collectin precursor, heat shock 27kDa protein, plasminogen activator inhibitor-1 precursor, thrombospondin 1 transitional endoplasmic reticulum ATPase 및 $\beta$-tubulin이었다.

• Clinical and Experimental Pediatrics
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• 제52권1호
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• pp.111-118
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• 2009

목 적 : 어린시기에 genistein과 같은 식물성 에스트로겐의 섭취가 사회적 관심사로 대두되고 있다. 본 연구에서는 어린 쥐에서 genistein에 노출이 사춘기 개시 및 생식기관에 미치는 영향을 알아 보았다. 방 법 : 이유기(3주령) 암컷 흰쥐를 저용량 genistein (10 mg/kg/day), 고용량 genistein (100 mg/kg/day), 대조군의 세 그룹 (각 그룹 당 n=6)으로 나누고 첫 번째 질구 개방이 확인되는 날까지 농도별로 각각 경구 투여하였다. 질구 개방일을 확인하고 생식 기관의 무게를 측정하며 난소와 자궁에서 $ER{\alpha}$, $ER{\beta}$, PR 유전자들의 발현양상을 RT-PCR을 이용해 비교하였고, 난소와 자궁의 구조적 이상을 확인하기 위해 조직학적 분석을 실시하였다. 결 과 : 고용량 genistein 투여군은 저용량군 및 대조군에 비해 질구 개방일이 유의하게 촉진되었다. RT-PCR결과, $ER{\alpha}$, $ER{\beta}$, PR의 전사활성은 genistein에 노출된 쥐들의 난소와 자궁에서 유의하게 증가하였다. 그라프 난포와 황체는 genistein 투여군의 난소에서만 발견되었고, 대조군의 난소에서는 1차, 2차 난포들과 작은 미성숙 난포들만이 관찰되었다. Genistein 처리군의 자궁에서도 내막층 근막층 및 상피층이 과다성장상태였으나 대조군에서는 모든 세포층과 분비선이 미약하게 발달하였다. 결 론 : 결론적으로, 사춘기 이전 시기에 비교적 단기간의 genistein 노출이라도 미성숙 암컷 흰쥐에서 생식 내분비 활성을 일으켜 조기 사춘기와 성 스테로이드 호르몬 수용체의 발현 양상 변화를 초래할 수 있으며, genistein의 노출이 아동기 성성숙에 미치는 영향에 대한 더욱 많은 연구가 필요할 것으로 사료된다.

• 대한수의학회지
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• 제34권4호
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• pp.881-890
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• 1994

Skim milk progesterone($P_4$) profiles in 74 dairy cows were determind to monitor postpartum ovarian activity by radioimmunoassay. Milk samples were collected from each cow every 5 days from 10 to 90 days postpartum. Signs of estrus were observed twice daily, and status of the ovaries and uterus were examined every 10 days by rectal palpation. Results are summarized as follows: 1. Cows were categorized into five types by the change of skim milk $P_4$ profiles; Type I(normal) : Cyclic changes of skim milk $P_4$ profiles appeared within 20 days postpartum(12 cows, 16.2%), Type II(cycle delayed) : Cyclic changes of skim milk $P_4$ profiles appeared from 21 to 60 days postpartum(39 cows, 52.7%), Type III(cycle ceased with low $P_4$) : Onset of the estrous cycle within 20 days postpartum but ceased later with low levels of $P_4$ (7cows, 9.5%), Type IV(cycle ceased with high $P_4$) : Onset of the estrous cycle within 20 days postpartum but ceased later with high levels(>3.0 ng/ml) of skim milk $P_4$ (4 cows, 5.4%), Type V(acyclicity) : Skim milk $P_4$ concentration remained low(<1.0 ng/ml) until 80 days postpartum(12 cows, 16.2%). 2. Out of the 17 cows classified as the Type III and Type V by skim milk $P_4$ profiles, 13 cows had inactive ovaries and remaining 6 cows had single or multiple follicular cysts in their ovaries by rectal palpation. All 4 cows of Type IV had a persistent corpus luteum in their ovaries. 3. Approximately eighty percent of the cows had begun ovarian activity by 60 days postpartum and 90.6% by 90 days by skim milk $P_4$ profiles, but only 39.2% by 60 days and 71.7% by 90 days had shown visible estrus signs. The mean days from parturition to the first, second and third ovulations determined by skim milk $P_4$ profiles was $28.0{\pm}11.0$, $46.4{\pm}13.3$ and $66.4{\pm}11.5$ days and the visible estrus signs were 9.3%, 38.1%, and 48.6%, respectively. The mean days from parturition to the first visible estrus was $57.2{\pm}15.9$ days. These results indicated that milk $P_4$ profile of each Types by radioimmunoassay can be utilized for monitoring postpartum ovarian and would be useful for the early detection of ovarian dysfunction in dairy cow.

• Clinical and Experimental Reproductive Medicine
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• 제27권4호
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• pp.393-403
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• 2000

Objective: To investigate the effects of ROS on kinematic parameters in human spermatozoa. To verify the changes in above parameters, human spermatozoa were incubated with xanthine-xanthine oxidase (X-XO), $H_2O_2$, sodium nitroprusside (SNP) or lymphocyte. Otherwise, spemlatozoa were incubated under low $O_2 (5%) condition. Methods: CASA was employed to analyze sperm motion parameters. Results: Under $H_2O_2 treatment, all kinematic parameters of spermatozoa were dramatically increased during 30 min, but gradually decreased thereafter. Under the low concentration of $H_2O_2 (125 ${\mu}M$ and 250 ${\mu}M$), the movement velocity (VAP, VCL, VSL) decreased, but forward movement increased. Under the 1mM $H_2O_2, sperm showed reduced kinematic parameters except during first 30 min of incubation. In the cases of X-XO and SNP treatment, the movement velocity increased but the forward movement reduced. After incubation for 3 hr treatment, the kinematic parameters gradually decreased in high concentration of X-XO. However these parameters maintained or increased in low concentration of X-XO. There was no obvious changes in the above parameters in the high concentration of SNP. In the presence of high concentration of lymphocytes, all parameters decreased. Under the 5% $O_2 condition, the parameters of the movement velocity and movement pattern increased, but forward movement decreased. Taken togethers, it suggested that ROS increased the movement velocity but decreased the forward movement and lateral head replacement. $H_2O_2, X-XO, SNP and lymphocyte treatment significantly increased capacitated spermatozoa within I h of incubation. There was no significant difference in capacitation between low- and high $O_2 group. Conclusion: The early onset of capacitation in the presence of ROS suggest that ROS might be a positive regulator of sperm capacitation and hyperactivation. These results demonstrate that low concentration ROS facilitates the movement velocity but high concentration ROS was inhibitory.

• Clinical and Experimental Reproductive Medicine
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• 제29권2호
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• pp.139-147
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• 2002

Objective : This study was to establish a reproducible differentiation system from the parthenogenetic mouse embryonic stem (P-mES02) cells into functional cardiomyocytes like as in vitro fertilization mouse embryonic stem (mES01) cells. Materials and Methods: To induce differentiation, P-mES02 cells were dissociated and aggregated in suspension culture environment for embryoid body (EB) formation. For differentiation into cardiomyocytes, day 4 EBs were treated with 0.75% dimethyl sulfoxide (DMSO) for another 4 days (4-/4+) and then were plated onto gelatin-coated dish. Cultured cells were observed daily using an inverted light microscope to determine the day of contraction onset and total duration of continuous contractile activity for each contracting focus. This frequency was compared with the results of DMSO not treated P-mES02 group (4-/4-) and mES01 groups (4-/4+ or 4-/4-). For confirm the generation of cardiomyocytes, beating cell masses were treated with trypsin-EDTA, dispersed cells were plated onto glass coverslips and incubated for 48 h. Attached cells were fixed using 4% paraformaldehyde and incubated with specific antibodies (Abs) to detect cardiomyocytes (anti-sarcomeric ? -actinin Ab, 1 : 100; anti-cardiac troponin I Ab, 1 : 2000) for 1 h. And the cells were finally treated with FITC or TRITC labelled 2nd Abs, respectively, then they were examined under fluorescence microscopy. Results: Rhythmically contracting areas in mES01 or P-mES02 cells were firstly appeared at 9 or 10 days after EBs plating, respectively. The highest cumulative frequency of beating EBs was not different in both treatment groups (mES01 and P-mES02, 4-/4+) with the results of 61.3 % at 13 days and 69.8% at 15 days, respectively. Also, the contracting duration of individual beating EBs was different from minimal 7 days to maximal 53 days. However, DMSO not treated groups (mES01 and P-mES02, 4-/4-) also had contracting characteristics although their frequency was a few compared to those of DMSO treated groups (6.0% and 4.0%). Cells recovered from the spontaneously contracting areas within EBs in both treated groups were stained positively with muscle specific anti-sarcomeric ? -actinin Ab and cardiac specific anti-cardiac troponin I Ab. Conclusion: This study demonstrated that the P-mES02 cell-derived cardiomyocytes displayed similarly structural properties to mES01 cell-derived cardiomyocytes and that the DMSO treatment enhanced the cardiomyocytes differentiation in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제26권1호
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• pp.75-81
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• 1999

The beneficial effect of glucose and phosphate ions in culture medium on the development of human embryos in vitro has not been fully elucidated. The purpose of this study was to evaluate the influence of fertilization and culture of embryos in glucose/phosphate-free m-TALP medium on pregnancy rates in IVF-ET program. The patients in 244 IVF-ET cycles received GnRH agonist + HMG regimens. A does of 10,000 IU HCG was administered when two or more dominent follicles reached 18mm in diameter. Thirty-six hours after HCG, oocytes were recovered transvaginally using ultrasound guidance. Aspirated oocytes were matured for 4 to 6 h in TCM-199 supplemented with 10% follicular fluid (FF). Insemination was carried out with 50,000 motile spermatozoa in TCM-199 + 10% FF or m-TALP + 5% FF + 5% fetal cord serum (FCS) according to experimental design. After 6 h, oocytes were washed 3 to 4 times and cultured in each fresh medium. After 20 h, oocytes were freed from cumulus/corona cells and examined for the presence of pronuclei. Fertilized oocytes were transferred into each co-culture drops and cultured for further incubation. On day 3, embryo transfer was performed with grade 1 and 2 embryos. Monolayers for co-culture of embryos were prepared by plating $1{\times}10^5$ cumulus cells/ml in 10ul drop of TCM-199 + 10% FF or m-TALP + 5% FF + 5% FCS media 24 h prior to the onset of co-culture. Development to 4 to 16 cell stage was observed at 70x magnification following two days of incubation. Pregnancy was confirmed by detecting increasing serum ${\beta}$-hCG concentrations for 11 days following embryo transfer. Data were analyzed by ${\chi}^2$-test. Oocytes from 244 IVF-ET cycles were randomized. The number of cycles and mean age of patients were 97 and 147, 31.3 yrs and 31.2 yrs for TCM-199 (control) and m-TALP groups, respectively. The mean number of retrieved oocytes/cycle, fertilization rates, number of embryos transferred/ET and pregnancy rates were 11.1 and 10.3, 65.1% and 67.3%, 4.1 and 4.7, 28.9% and 43.8% for TCM-199 and m-TALP groups, respectively. Differences in the pregnancy rates were found between control and m-TALP groups (p<0.05). The pregnancy rate of patients divided according to maternal age groups of ${\leq}30$, 31-35, $36{\leq}$ were 44.4% and 49.0%, 26.1% and 41.3%, 29.2% and 41.2% for control and m-TALP groups, respectively. These data indicate that culture of human embryos in glucose/phosphate-free m-TALP medium improves pregnancy rates.

• 한국동물생명공학회지
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• 제35권4호
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• pp.329-337
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• 2020

Cytochrome P450 1A2 (CYP1A2) is a member of the cytochrome P450 superfamily enzymes in mammals and plays a major role in metabolizing endogenous hormones in the liver. In recent days, CYP1A2 expression has been found in not only the liver but also other tissues including the pancreas and lung. However, little information is available regarding the expression of CYP1A2 in the ovary, in spite of the facts that the ovarian follicle growth and atresia are tightly associated with controls of endocrine hormonal networks. Therefore, the expression of CYP1A2 in the ovaries of prepubertal and pubertal rats was investigated to assess its expression pattern and puberty-related alteration. It was demonstrated that the expression level of CYP1A2 was significantly (p < 0.01) higher in the pubertal ovaries than prepubertal counterparts. At the ovarian follicle level in both groups, whereas CYP1A2 expression was less detectable in the primordial, primary and secondary follicles, the strongly positive expression of CYP1A2 was localized in the granulosa cell layers in the antral and pre-ovulatory follicles. However, the ratio of CYP1A2-positive ovarian follicle was significantly (p < 0.01) higher in the ovary of pubertal group (73.1 ± 3.1%) than prepubertal one (41.0 ± 10.5%). During the Immunofluorescence, expression of CYP1A2 was mainly localized in Fas-positive follicles, indicating the atretic follicles. In conclusion, these results suggested that CYP1A2 expression was mainly localized at the atretic follicular cells and affected by the onset of puberty. Further study is still necessary but we hypothesize that CYP1A2 expresses in the atretic follicles to metabolize residue of the reproductive hormones. These findings may have important implications for the fields of reproductive biology of animals.

• 한국수정란이식학회지
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• 제28권2호
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• pp.149-155
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• 2013

Mullerian inhibiting substance (MIS) is a member of the TGF-${\beta}$ (transforming growth factor-${\beta}$) family whose members play key roles in development, suppression of tumour growth, and feedback control of the pituitary-gonadal hormone axis. MIS is expressed in a highly tissue-specific manner in which it is restricted to male Sertoli cells and female granulose cells. The serum levels of MIS in prenatal and postnatal ICR mice were measured using the enzyme-linked immuno-solvent assay (ELISA) using the MIS/AMH antibody. Mice were grouped by age: the significant periods were at the onset of development. During sex organ differentiation, no remarkable difference between female and male foetus MIS serum levels (both<0.1 ng/ml) was observed. However, MIS serum levels in pregnant mice markedly changed (4.5~12.2 ng/ml). After birth, postnatal female and male mice serum MIS levels changed considerably (male: <0.1~138.5 ng/ml, female: 5.3~103.4 ng/ml), and the changing phase were diametrically opposed (male: decreasing, female: fluctuating). These findings suggest that MIS may have strong associations with not only develop-ment but also puberty. For further studies, establishing the standard MIS serum levels is of importance. Our study provides the basic information for the study of MIS interactions with reproductive organ disability, cancer, and the effect of other hormone or menopause. We hypothesise that if MIS is regularly injected into middle-age women, meno-pause will be delayed. We detected that serum MIS concentration curves change with age. The changing phase is different between males and females, and this difference is significant after birth. Moreover, MIS mRNA is expressed during the developmental period (prenatal) and also in the postnatal period. This finding indicates that MIS may play a significant role in the developmental stage and in growth after birth.

• 한국수정란이식학회지
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• 제31권4호
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• pp.349-353
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• 2016

This study was carried out to evaluate the influencing factors that affect the reproductive performance of cows at the Monirampur upazila in Jessore district of Bangladesh. A total of 224 cows were brought to the upazila livestock hospital for artificial insemination (AI). The cows were inseminated between 12 to 18 hours from the onset of estrus and data was obtained from the owner. Out of 224 cows, 133 became pregnant and 91 were non pregnant. In this study, the overall pregnancy rate was 59.29%. Among the age variability, the highest pregnancy rate (70.27%) was at the age of 4 years old. In case of breed variation, the highest pregnancy rate was observed in local breed (69.07%) compared with other crossbred cows. Hence the breed variations significantly influence the conception rate of cows. According to the parity, we found that the pregnancy rate was increasing with their parity but decreasing after 4th parity. The highest conception rate was observed in 3rd parity (67.74%) which was significantly higher than that of heifers (Parity-0). Here we also found that the types of bull semen used for AI had no significant effect for pregnancy rate. The skills of AI technician for AI to cows were significantly affecting the pregnancy rate. However, this study is not enough for rating and comment about the reproduction performance of cows. Therefore, further extensive study is needed for rating and recommendation for the cattle up gradation at that particular area.

• 한국동물생명공학회지
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• 제35권2호
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• pp.171-176
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• 2020

Advancing the estrous cycle of mares is an essential breeding strategy that is routinely conducted by Thoroughbred breeders to improve economic outcomes. For this purpose, Equilume light masks have been developed as an alternative to existing technologies such as artificial lighting or hormonal treatments because they are considered as valid as existing methods with additional animal welfare advantages. For example, with the Equilume light masks, horses can be let out into the pasture, whereas they have to be kept indoors during lighting treatment. Because the function of Equilume light mask on the estrous cycle of mares is influenced by environmental factors such as nutrition condition and temperature, Equilume light mask should be studied in various environments. The objective of the present study was thus to verify the effect of Equilume light masks on the onset of the estrous cycle in Thoroughbred mares in South Korea. Mares were randomly selected and separated into two groups at two Thoroughbred horse breeding farms. The mares in the treatment group were equipped with Equilume light masks from November 18 to February 10 the following year. The body condition, the number and size (> 35 mm) of uterine follicles, and the uterine horn score of the mares were assessed on January 6 and February 10. The body condition scores were not different between the two groups. The treatment with the Equilume light mask had no positive effects on developing follicles and the reproductive tract. In conclusion, the use of Equilume light masks did not influence the seasonal resumption of the estrus cycles in Thoroughbred mares in South Korea.