• Clinical and Experimental Reproductive Medicine
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• 제27권1호
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• pp.23-29
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• 2000

Objective: Zona pellucida (ZP) has been thought to be the barrier of egg to sperm penetration before and after fertilization. The phenomenon of ZP hardening has been considered as a post-fertilization event until now, and it is generally accepted that it is caused by the secretory products of cortical granules released during the cortical reaction. Hardening of ZP could occur "spontaneously" in mammalian oocytes in standard culture conditions, and that it is probably not a consequence of cortical reaction. The purpose of our study was to investigate the effect of human amniotic fluid (HAF) on nuclear maturation (NM) and fertilization ability of mouse immature oocytes. Methods: HAF was obtained from patients undergoing amniocentesis at $16{\sim}20$ weeks of gestation. HAF from five to ten patients was centrifuged and the supernatants was pooled. Cumulusenclosed mouse immature oocytes were incubated in the medium containing HAF, and examined to confirm NM and fertilization. Female ICR mice (about 3 weeks old) were stimulated with 7.5 IU PMSG. Immature oocytes were isolated at $48{\sim}52$ hrs post PMSG injection and cultured in TCM-199 supplemented with 20% HAF for 18 hrs. FBS was used as a control for the examination. Matured oocytes (MII) were fertilized with sperms collected from the epididymis of male mice (over 10 weeks old). Fertilization was in conducted T6 medium containing 15 mg/ml BSA, and confirmed at 6 hrs post-insemination. Fertilization rate was assessed in zona-intact or zona-free oocytes (denuded by trypsin). Evaluation of NM and fertilization was carried out by rapid staining method. ZP hardening was evaluated by incubating cumulus cell-free mature oocytes in 0.001% chymotrypsin at $37^{\circ}C$ for 10 min. Results: There was no significant difference between the effects of HAF (86.6%) and FBS (87.7%) supplements on NM of immature oocytes. When maturation medium was supplemented with HAF, total fertilization rates (7%) were significantly lower (p<0.01) than that of FBS (85.1%). In HAF group, fertilization rate was increased (p<0.01) in zona-free oocytes (7% versus 100%). The resistance of mouse oocyte ZP to digestion by chymotrypsin after maturation in vitro was significantly higher (p<0.01) in HAF group (86.7%) than in FBS (6.7%). To culture oocytes in FBS were very effective in preventing ZP hardening. However cultured oocytes in HAF showed high rate of ZP hardening (p<0.01). Conclusions: These results suggest that HAF can be used as a supplement for the NM of mouse immature oocytes in vitro. However, HAF induces spontaneous hardening of ZP of mouse immaure oocytes during maturation in vitro.

• Clinical and Experimental Reproductive Medicine
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• 제29권1호
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• pp.21-28
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• 2002

Objective: To evaluate whether co-culture of oocytes on vero cell monolayers from Day 0 (Day 0 group) after egg retrieval results in an increase in developmental capacity such as fertilization rate, embryo quality, blastulation and clinical pregnancy rate compared with co-culture of oocytes from Day 1 (Day 1 group). Methods: Sperms were treated with Hams F-10 supplemented with 10% human follicular fluid (hFF). Vero cells for co-culture were prepared in TCM-199 with 10% FBS. Oocytes were co-cultured from Day 0 and fertilized oocytes were co-cultured from Day 1 on vero cell monolayers in DMEM with 10% and 20% hFF, respectively after egg retrieval. On day 1, 2 and 5, fertilization rate and grade of embryos and blastocysts were evaluated. Results (fertilization rate, cleavage rate, grade of embryos and blastocysts and pregnancy rate) were considered statistically significant when p value was less than 0.05 using t-test and $x^2$. Results: In sibling oocytes of same cycles, no differences were found in fertilization rate (94.6 vs. 91.4%), cleavage rates (94.6 vs. 91.4%), embryo grade (on day 2 and 3) and blastulation (65.6 vs. 57.0%) and their grade. In different oocytes of different cycles (patients), no differences were found in fertilization (79.8 vs. 78.3%), cleavage rates (77.7 vs. 76.4%) and blastulation (56.0 vs. 45.3%), but pregnancy rate was higher in the Day 0 group than in the Day 1 group (60.0 vs. 42.9%). Conclusions: This study revealed that the embryonic development capacities were not affected by the different co-culture time in the sibling oocytes of same cycles. Although no statistical significance, because of small size of study, there was a trend for higher pregnancy rates in Day 0 group compared to Day 1 group in different oocytes of different cycles.

• Archives of Pharmacal Research
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• 제25권3호
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• pp.357-363
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• 2002

The effect of a new rhFSH, PG-0801, on oocyte quality, ovulation and in vitro fertilization (IVF) was examined in androgen-sterilized mice. Experimental sterility was induced by a single subcutaneous injection of testosterone propionate (TP, 1 mg/head) into 5 day old female mice. Ovulation was generated in the 10 to 13-week old TP-injected mice by a subcutaneous rhFSH injection (1, 5 or 10 IU/head) followed 48 hours later by a second rhFSH injection (1, 5 or 10 IU/head). For comparison, a subcutaneous PMSG (5 IU/head) injection was used for folliculogenesis and a hCG (5 IU/head) injection was used for ovulation. These were administered using the same protocol. The eggs were harvested from the oviducts and counted 17 to 20 hours after the second injection. IVF was performed by adding sperms ($2{\times}10^{5}/ml{\;}to{\;}2{\times}10^{6}/ml$) to determine the functional activity of the eggs, and the fertilization rate was measured. In addition, the pregnancy rate and fetal development were examined after 15-17 days of gestation. The number of oocytes recovered from the rhFSH/rhFSH group increased dose-dependently and was slightly higher than that of the PMSG/hCG group. The pregnancy rates of the group receiving 1, 5, and 10 IU of rhFSH/rhFSH were 50%, 66.7%, and 75%, respectively, which were significantly higher than that of the control (untreated) group (0%). The numbers of viable fetuses in the 1, 5, and 10 IU/head of the rhFSH/rhFSH group ($8.0{\pm}1.50$, $8.9{\pm}1.02$, and $8.9{\pm}1.12$ fetuses/dam, respectively) were comparable to that of the 5 IU/head PMSG/hCG group ($9.4{\pm}0.94$). The mice receiving rhFSH/rhFSH and PMSG/hCG showed similar fertilization rates (around 65%) via the IVF procedure. These results demonstrate that a new rhFSH, PG-0801, may be useful for inducing ovulation in functionally infertile patients and for superovulation in ovulatory patients participating in assisted reproductive technology (ART) programs.

• Parasites, Hosts and Diseases
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• 제36권4호
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• pp.217-225
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• 1998

미야타흡충 (Metagonimus miyatai) 피낭유충 및 성충의 표피 미세구조를 주자전자현미경으로 관찰하였다. 피낭유충은 피라미 (Zacco platypus)의 비늘에서 분리하였고, 성충은 흰쥐에 피낭유충을 감염시킨 후 1-4주에 소장으로부터 회수하였다. 탈낭한 피낭유충의 전체적인 외형은 요코가와흡충과 큰 차이가 없었으며, 복흡반은 충체 중앙으로부터 오른쪽에 위치하였다. 구흡반의 구순에는 제I형 감각유두 및 제II형 감각유두가 여러 개 관찰되었다. 복흡반 앞쪽에는 끝이 5-7분지된 피극들이 빽빽하게 덮혀 있었으며, 충체 후반부의 배설공 부근에는 2-3분지된 피극으로 덮혀 있었다. 감염 1주된 성충의 경우, 구흡반의 구순에는 제II형 감각유두 7개가 분포하고 있었으며, 구흡반 내벽에는 큰 제I형 감각유두가 1쌍씩, 작은 제I형 감각유두가 2쌍씩 좌우측에서 각각 관찰되었다. 피극의 분포는 피낭유충의 경우와 비슷하였으나, 분화도는 증가하여 9-11분지된 피극들이 관찰되었다. 감염 2-4주된 성충의 표피 미세구조는 감염 1주된 성충과 대동소이하였으나, 정자가 충체 배측 표면으로 개구된 Laurer＇s canal로 들어가고 있는 것이 관찰되었다. 이상의 결과를 종합할 때, 미야타흡충의 표피 미세구조는 요코가와흡충과 전반적으로 비슷하였으나 피극의 분포 및 분화도, 구흡반 주변의 감각유두 분포 등에 다소 차이가 있음을 알 수 있었으며 이러한 차이가 분류학적으로 의미있는 것일 가능성이 제시되었다.

• 한국발생생물학회지:발생과생식
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• 제13권4호
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• pp.321-327
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• 2009

망간은 정소 독성을 나타내며, 뇌기저핵에 작용하여 혈청 프로락틴의 농도를 증가시킨다. 그리고 혈청 프로락틴 농도 상승에 의한 과프로락틴혈증(hyperprolactinemia)은 정소의 정자 생성을 억제한다. 본 연구에서는 망간의 전신 노출이 흰쥐 정소의 정자 생산과 혈청 프로락틴 농도에 미치는 영향을 조사하기 위하여 실험동물을 대조군 $(0.0mg/m^3)$과 망간 노출군 (Mn $1.5mg/m^3$)으로 나누고, 노출군은 다시 노출 기간에 따라 4주와 13주 노출군 등 4군으로 분류하였다(n=10). 노출 기간에 따라 실험동물의 체중 변화와 사료 섭취량 등 일반적 소견 관찰, 혈액과 정소의 망간 농도, 정자의 수와 기형 등을 관찰하였다. 그리고 망간 노출에 따른 혈청 프로락틴 농도를 조사하여 망간 노출 조건에 따른 혈청 프로락틴 농도 변화 및 정소 독성을 조사하였다. 망간 노출 4주 및 13주군에서 노출기간에 따라 혈액 및 정소의 망간 농도가 유의하게 증가되었다. 대조군에 비하여 망간 노출군에서 노출기간에 따라 정자의 수가 감소되었으며, small head와 bent tail 등 기형 정자의 빈도는 증가하였다. 혈청 프로락틴의 농도는 망간 투여군에서 대조군에 비하여 유의하게 증가하였다. 그러나 실험동물의 체중 변화 및 사료 섭취량은 실험군간에 차이가 없었다. 이러한 결과들로 보아 $1.5mg/m^3$ 농도의 아만성 망간 노출은 흰쥐의 혈청 프로락틴 농도를 증가시키고, 정소 독성의 원인으로 추정된다. 그리고 전신 노출에 의한 망간의 흰쥐 정소 독성의 무유해영향농도(NOAEL)는 $1.5mg/m^3$ 이하로 예측된다.

• Reproductive and Developmental Biology
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• 제35권4호
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• pp.479-483
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• 2011

The objective of this study was to determine the effects of E. coli isolated from porcine semen on sperm viability, motility, and semen pH. Semen samples were prepared using commercial extender, $Seminark^{Pro}$ (Noahbio Tech, Korea) that did not contain antibiotics. And 4 different levels of E. coli were artificially innoculated to semen with following concentrations; 4,000 of sperms with 1 of E. coli (T1), 400 with 1 (T2), 40 with 1 (T3), and 4 with 1 (T4). Semen samples were preserved at $17^{\circ}C$ for 5 days in semen storage box until analyzed by flowcytometer. Aliquots were subjected to measure the sperm viability (Live/$Dead^{(R)}$ stain), motility (mitochondrial function), and semen acidity (pH) from day 0 (day of semen collection) to day 5. Sperm motility and viability were significantly decreased (p<0.05) on day 0 (4 hrs after preservation at $17^{\circ}C$) in T3 and T4 compared to control groups and were significantly decreased (p<0.05) in all groups from day 3. Sample pH was acidic in T3 (6.90~6.86) and T4 (6.86~6.65) from day 3 to day 5 (p<0.05). On the other hand, sample pH was maintained 7.0~7.1 in control, T1, and T2 during the experimental period. Sperm motility and viability were significantly decreased from day 0 to day 5 compared to control in samples contaminated with E. coli above a value of 40:1 ($20{\times}10^6$ sperm cells/ml : $5{\times}10^5$ cfu/ml). Even on day 1 in T4 and on day 3 in T3, semen pH was acidic probably due to the acidification of dead spermatozoa. These results suggest that E. coli contamination has a concentration-dependent detrimental effect on extended porcine semen quality.

• 한국가축번식학회지
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• 제8권1호
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• pp.46-55
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• 1984

The purpose of the experiment was to clarify morphologically normal growth pattern of the ductus deference in accordance with the sex maturity of meat-type cockerels. 1. Diameter of lumens in u, pp.r, mid and lower parts of ductus deferens, the most conspicuous enlargement of lumen was observed in the lower part. Heights of epithelial layers of ductus deferens showed abrupt growth at 12 weeks of age with subsequent gradual growth in all the part of u, pp.r, mid and lower, and heights of those at 30 weeks were a, pp.oximately 4 times as large in the u, pp.r and mid parts and 5 times as large in the lower part in contrast to those at 4 weeks of age. Thickness of muscular layer of ductus showed gradual growth in contrast with the diameter of lumen and height of epithelial layer, showing 1.3 times as large in the u, pp.r part, 1.6 times in the mid part and 1.9 times in the lower part at 30 weeks of age in contrast to the thickness at 4 weeks of age. 2. Within 10 weeks after hatching, lining cells of ductus deferens were mainly composed of round cells and columnar cells in simple columnar epithelium. During 10th to 20th week, the lining cells were mainly composed of high columnar cells and round cells in pseudostratified epithelium. From 22nd week, the lining cells were composed of pseudostratified columnar cells. Whereas round cells disa, pp.ared gradually. Enlargement of lumen and pooling of sperms in ductus deferens coincided with the maturation of seminiferous tubules. 3. In simple correlation between the values of testis weight and the values from various measurements in the ductus deferens, there was significant correlation coefficient with each other. 4. In the India ink absorption test, India ink granules were not absorbed on the epithelium of the ductus deferens, but the granules reactive to acid phosphatase a, pp.ared in a line on the free border of each parts of the ductus deferens. The granules reactive to alkaline phosphatase were noted on the luminal border of ductus deferens mainly, but weak reaction showed than acid phophatase were a, pp.ared. The granules reactive to PAS were a, pp.ared mostly near on the free border of hte epithelial cells of ductus deferens. 5. Number of sperm, Indes of sperm vitality and MRT in the different parts of ductus deferens were tended to be somewhat dominant in the mid and lower parts than in u, pp.r part, even though not significant in the statistical analysis. Ratio of sperm abnormality was tended to be relatively high in the u, pp.r part too, and in the sperm of abnormality blunted head was less in number significantly in the mid and lower part than in the u, pp.r part.

• 한국가축번식학회지
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• 제25권2호
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• pp.147-153
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• 2001

본 연구는 Pb중독이 rat의 생리현상에 미치는 영향을 구명 하고자 연속적인 Pb acetate 투여가 정소의 중량, 정자수, 활력, 장기중량 및 조직학적 변화를 조사하였다. 1. Pb acetate 1,000, 2,000 및 4,000 ppm/kg을 rat에 투여했을 때 정소중량은 정상대조군에 비해 감소하는 경향을 나타냈고 용량이 증가할수록 정소중량은 큰 감소경향을 나타냈다. 2. Pb acetate 1,000, 2,000 및 4,000 ppm/kg을 rat에 투여했을 때 정자수는 정상대조군에 비해 감소하는 경향을 나타냈고 용량이 증가할수록 정자수는 유의한 감소경향을 나타냈다. 3. Pb acetate 1,000, 2,000 및 4,000 ppm/kg을 rat에 투여했을 때 정자의 활력은 정상대조군에 비해 점차 감소하는 경향을 나타냈고 용량이 증가할수록 정자활력은 큰 감소경향을 나타냈다. 4. Pb acetate 1,000, 2,000 및 4,000 ppm/kg을 rat에 투여했을 때 간 및 신장의 중량은 정상대조군에 비해 점차 증감의 경향을 나타냈으며, 투여용량별 간 및 신장의 중량은 유의한 변화를 나타내지 않았다. 5. Pb acetate를 연속적으로 투여했을 때 간의 조직학적 관찰소견은 간세포 중심정맥의 주위성괴사, 호중구의 침윤, 담즙정체 및 간문맥관과 간질 대식세포의 침윤이 관찰되었고, 신장에서는 원위세뇨관 상피의 핵내 함입물과 사구체낭 및 세뇨관내 균질한 초자물질이 충만되어 있었고 림프절에서는 골수의 조혈현상이 관찰되었다.

• Reproductive and Developmental Biology
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• 제40권3호
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• pp.27-31
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• 2016

The aim of this study was to evaluate effect of ${\alpha}$-linolenic acid (ALA) on viability, acrosome reaction and mitochondrial intact in frozen-thawed boar sperm. The boar semen was collected by gloved-hand method and cryopreserved in 20% egg yolk freezing extender containing ALA (0, 3, 5, and 10 ng/mL) with 0.05% ethanol. The frozen-boar spermatozoa were thawed at $37.5^{\circ}C$ for 45 sec in water-bath. The spermatozoa samples were evaluated the plasma membrane integrity, acrosome reaction, and mitochondrial integrity using flow cytometry. In results, population of live sperm with intact plasma membrane was significantly higher in control and 3 ng/mL ALA treatment group than ethanol group (p<0.05). In contract, dying sperms were higher in ethanol group than 3 ng/mL ALA treatment (p<0.05). Acrosomal membrane damage in all sperm population was reduced in 3 ng/mL ALA groups compared with ethanol treatment (p<0.05). However, acrosome damage in live sperm population was no significant difference among the all treatment groups. Mitochondrial integrity was not influenced by ALA treatments in both of live and all sperm population. In conclusion, this results show that supplement of ALA during the cryopreservation process could reduce the membrane damages including plasma and acrosomal membrane, whereas ALA did not influence to mitochondria in boar spermatozoa. Therefore, these results suggest that ALA can protect against the membrane damage derived cryo-stress, and cryopreservation efficiency of boar semen would be improved by use of ALA.

• 한국수정란이식학회지
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• 제25권4호
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• pp.229-235
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• 2010

The MTT assay is one of superior evaluation methods widely used to analyze the viability of metabolically active cell. It can be used to determine the percentage of viable sperm through measurement of the reduction of MTT granules at mitochondria in sperm tail. The purpose of this study is to determine the optimal condition of a simple and easy MTT assay to validate boar sperm viability and compare the accuracy of this test with microscopic examination. The MTT reduction rate for sperm viability were analyzed in microtiter plates (96 well) from 1 hr to 5 hr incubation periods at $37^{\circ}C$ using spectrophotometer (microplate reader) at 550 nm wavelength. The remainder of semen sample was simultaneously examined to compare the correlation of accuracy between MTT assay and other sperm parameters. Those sperm parameters were included the motility, survival rates, membrane integrity, mitochondria activity and acrosome integrity. The OD values of MTT assay (MTT reduction rates) did not greatly change at 1 hr to 5 hr incubation periods in different proportion of live and freeze-killed sperms (dead sperm). The MTT reduction rates or survival rates were decreased according to the different concentration of live and dead sperm. The linear regression at 1 hr and 4 hr incubation periods in sperm MTT assay was y=291.55x-72.176 and y= 180.64x-44.569, respectively. There are high correlation between 1 hr and 4 hr incubation periods (p<0.001). The results of MTT assay and other sperm parameters has a positive correlation (p<0.01 or 0.05). The correlation coefficients for MTT assay was 0.88115 for motility, 0.89868 for survival rates, 0.91722 for membrane integrity and 0.77372 for acrosome integrity, respectively. In conclusion, the MTT assay can be used as a reliable and efficient evaluation method for boar sperm viability. It can be use practical means to evaluate the quality of boar sperm by a fast, inexpensive and easy method.