• 한국발생생물학회지:발생과생식
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• 제4권2호
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• pp.181-186
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• 2000

생쥐 난소에서 glucosamine-6-phosphate deaminase (GNPDA)의 발현을 조사하였다. Western blot상에서 Mr. 31 kDa의 항원을 검출하였으며 출생 후2주에 급격한 발현의 증가가 확인되었다. 난소절편의 면역염색 결과 협막세포와 간충조직에서는 균질한 발현을 보인 반면 난포내 발현양상은 난포의 발달에 따라 다르게 나타났다. 일부 1차 난포의 난자에서 GNPDA의 발현이 관찰되었으나 강소형성 난포의 난자에서는 관찰되지 않았다. 황체화 과립세포에서의 발현은 황체발달에 따라 증가하였으며 황체퇴화 부위에서 강한 신호가 검출되었다. 난포발달에 따른 GNPDA발현의 차이는 GNPDA가 생쥐 난소 조직 재구성에 관여하고 있음을 암시한다.

• Journal of Radiation Protection and Research
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• 제23권2호
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• pp.89-96
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• 1998

뇌하수체 분비호르몬인 FSH의 방사선방어 효능을 조사하고자 생후 3주된 미성숙 생쥐에 $\gamma$ 선을 전신조사하거나(R), 전신조사 후 FSH 5 IU를 복강주사(RF)하였다. 대조군은 동량의 식염수를 복강주사하거나(C) 혹은 FSH를 복강주사(F)하였다. 0시간, 6시간, 12시간 1일, 2일, 4일, 그리고 8일 후에 각 군별로 5마리씩 경추파열로 도살한 후 난소를 채취하였다. 난소는 neutral buffered formalin에 고정한 후 통상적인 표본조직을 만들었다. HE 염색을 기본으로 면역조직화학염색을 비교하였으며 전체 DNA를 추출하여 agarose gel 전기영동을 실시하여 DNA fragmentation의 정도를 알아보았다. 방사선 조사군인 R군과 RF군에 있어서 6시간에서 12시간에 apoptosis를 반영하는 면역조직화학적 염색률이 높게 나타났으며, 8일 경과시 분열세포의 존재비를 반영하는 염색률이 높게 나타났다. 또한 DNA fragmentation의 정도를 보면, 모든 실험군에서 185bp의 DNA ladder가 시간에 따라 증가하는 경향을 보였다. 370bp의 DNA는 R군의 경우 6시간에 나타났으며 1일 이후 감소하였다. RF군의 경우 12시간에 나타나 1일 이후 감소하였다. F군에서는 370bp가 보이지 않았다. 본 실험의 결과, 강소형성 난포가 피폭되는 경우 4일 이후 8일에 이르러 난소내에서 완전히 소멸되고, 8일에 새로운 원시난포가 성장하는 것이 확인되었다 FSH는 방사선에 피폭된 난포의 퇴화를 지연 혹은 억제시키는 방사선방어 효과가 있었으며, 방사선에 의한 난포의 퇴화는 과립세포의 apoptosis를 매개로 하여 일어남을 알 수 있었다.

• 한국가축번식학회지
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• 제17권4호
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• pp.279-285
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• 1994

The objective of the present study was to characterize the cell cycles of granulosa cell populations during follicular maturation in cattle by using flow cytometer. Granulosa cells were isolated from bovine preovulatory antral follicles of F1(>10mm), F2(5~20mm), F3(3~4mm) and F4(1~2mm) diameter and fixed and stained with fluorochromes that selectively bine to DNA. Flow cytometer equipped with a laser excitation system was used to analyze the intensity of fluorescence from stained cells. Forward angle light-scatter(FSC) and 90$^{\circ}$light-scatter(SSC) signals were adopted to measure the size and the granularity of granulosa cells. As a results of FSC/SSC analysis, granulosa cell populations(G1 phase of cell cycle) from each follicle were relatively regular in size and granularity, regardless of follicular size. However, their distribution in granularity was greater than that in size. Most of granulosa cell populations collected from each follicle were distributed in G0/G1, S and G2/M phases. As the follicles approached to ovulation the percentage of cells in the proliferative phases of cell cycle (S and G2/M) decreased significantly, but there was a concomitant increase in the percentage of granulosa cells in G1 phase. Therefore, these data indicate the proportion of main populations to cell cycle of granulosa cells may be changed from proliferative phase to G1 phase during follicular maturation in cattle.

• Clinical and Experimental Reproductive Medicine
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• 제30권1호
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• pp.47-55
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• 2003

Objectives: Bok, Bcl-2-related ovarian killer, is a proapoptotic Bcl-2 family protein identified in the ovary based on its dimerization with the antiapoptotic protein Mcl-1. The present study examined the hormonal regulation and localization of Bok messenger RNA levels in the mouse ovary during the follicle development. Methods: The animals were implanted subcutaneously with Silastic brand capsules containing the synthetic estrogen, DES at $21{\sim}23$ days of age. Ovaries were collected $1{\sim}3$ days after implantation for RNA analysis and in situ hybridization. Some mice were removed capsule for $1{\sim}2$ days to induce ovarian follicle apoptosis. Ovaries were also collected from 26 day-old immature mice at various times after treatment with 10 IU PMSG. Some mice received a single intraperitoneal injection of 10 IU hCG to induce ovulation, and ovaries were obtained at different time intervals for Northern blot and in situ hybridization analysis, respectively. Results: Treatment of immature mice with diethylstilbestrol (DES) for $24{\sim}48$ h increased ovarian Bok mRNA levels. Bok mRNA was remained the same levels in mice removed DES for $24{\sim}48$ h to induce apoptosis. High signals of Bok mRNA after DES treatment were detected in granulosa cells of early antral follicles. Treatment of immature mice with PMSG for 12 h increased markedly ovarian Bok mRNA expression which was detected mainly in preantral and atretic follicles. Interestingly, low levels of Bok mRNA were also expressed in granulosa cells of preovulatory follicles. Treatment of PMSGprimed mice with hCG stimulated strongly ovarian Bok mRNA expression at $6{\sim}9$ h. At that time, Bok mRNA was expressed in granulosa cells of atretic and small growing follicles. Conclusion: These results demonstrate that Bok is one of proapoptotic Bcl-2 members expressed in early growing and atretic follicles during the ovarian follicular development. Gonadotropins induce a transient increase of Bok gene expression in granulosa cells of preantral and preovulatory follicles indicating some role in the ovulatory process.

• 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.

• Clinical and Experimental Reproductive Medicine
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• 제12권2호
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• pp.71-79
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• 1985

Systemic extrogen therapy promotes multiple preantral follicular development in immature mice. Estrogen pretreated ovaries might therefore be a useful source of cells for in vitro studies of oocytes maturation. Silastic capsules (5.0 mm length; 3.18 mm outer diameter, 1.57 mm inner diameter) filled with diethylstilbesterol were implanted subcutaneously in experimental mice (ICR) for up to 6 days. Ovarian weight and histology in diethylstilbesterol pretreated and control animal were assessed before and after pregnant mare serum gonadotrophin treatment and after human chorionic gonadotrophin. The following results were obtained; 1. Ovarian weight was significantly increased by 6 days of diethylstilbesterol pretreatment. Subsequent ovarian weight gain in response to pregnant mare serum gonadotrophin and human chorionic gonadotrophin was increased. 2. Diethylstilnbesterol pretreatment stimulated the developed healthy preantral follicles. 3. Forty eight hours after pregnant mare serum gonadotrophin treatment, a larger number of the antral follicles which developed in diethylstilbesterol pretreated animals showed signs of atresia, whereas in the control ovaries there was a higher incidence of premature luteinization. 4. Forty eight hours after human chorionic gonadotrophin, numerous corpora lutea and occasional luteinized unruptured follicles were present in both control and diethylstilbesterol ovaries. 5. Ovulation rate, fertilization rate and subsequent preimplantation development in vitro were not adversely affected by diethylstilbesterol pretreatment. However, there was considerable variation in the ovulation rate the number of animals with more than 60 ovulations was greater in the diethylstilbesterol gorup (52.4%) as compared to the control (33.3%).

• Asian-Australasian Journal of Animal Sciences
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• 제16권7호
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• pp.1071-1086
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• 2003

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.

• Asian-Australasian Journal of Animal Sciences
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• 제14권9호
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• pp.1342-1351
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• 2001

Over the years, the embryo transfer industry has grown from the simple collection & transfer of embryos into an advanced field of embryo biotechnology. Currently a large demand exists for bovine oocytes and early embryos in both research and commercial settings. Bovine embryos can now be produced in-vitro. Primary oocytes collected from antral follicles of abattoir - obtained ovaries can be induced to undergo the maturation process. In-vitor maturation system, however must ensure that the resulting oocyte is capable of undergoing normal fertilization and yields a zygote competent of developing to term after embryo transfer. Sperm preparation for IVF has improved with the use of heparine. The use of co-culture system has proved beneficial in circumventing the developmental block in IVM/IVF bovine embryos.

• Asian-Australasian Journal of Animal Sciences
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• 제11권5호
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• pp.480-485
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• 1998

Application of trans rectal ultrasonography to buffaloes (n=6) to follow the growth of large antral follicles individually, on each day of one interovulatory interval revealed that follicular turnover during oestrous cycle occured in waves. There was a predominance of a two-wave pattern (5/6 animals) compared to that of a three-wave pattern (1/6 animals). For two-wave pattern, the first wave emerged at Day $0.20{\pm}0.19$ (Day 0 = day of ovulation) and was marked by development of a dominant anovulatory follicle which grew in size from $5.40{\pm}0.24mm$ at the day of detection to a maximum diameter of $12.40{\pm}0.81mm$ on Day $8.60{\pm}1.57$, with a growth rate of $0.88{\pm}0.17mm/day$ and then regressed, with a mean persistence of $19.40{\pm}1.54$ days. The second wave emerged at Day $9.20{\pm}1.06$ and was marked by development of a dominant ovulatory follicle which grew in size from $4.20{\pm}0.37mm$ at the day of detection to a maximum diameter of $13.80{\pm}0.37mm$ on Day $21.00{\pm}1.38$, with a growth rate of $0.66{\pm}0.12mm/day$ and then ovulated on Day $21.60{\pm}1.25$, with a mean persistence of $11.80{\pm}1.39$ days. The maximum diameters attained and the growth rates of dominant anovulatory and dominant ovulatory follicles, and the mean number of follicles ${\geq}3mm$ diameter detected at the time of emergence of first and second waves ($11.80{\pm}1.74$ and $9.00{\pm}2.81$, respectively) were not significantly different. In the animal which showed a three-wave pattern, the first, second and third waves emerged on Days 1, 10 and 19, respectively. All animals, except one had at least one subordinate follicle in the first or second or both waves. The subordinate follicles increased in diameter over a few days and then regressed. The results indicate that in buffaloes, the follicular turnover during oestrous cycle occurs predominantly in a two-wave pattern.

• Journal of Radiation Protection and Research
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• 제19권3호
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• pp.179-188
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• 1994

생쥐의 난소에 미치는 방사선의 조직학적 및 기능적 영향을 조사하기 위하여 2.88Gy 및 7.2Gy의 방사선(Co-60)을 전신조사하였다. $7{\mu}m$ 두께의 조직절편을 제작하여 조직학적 관찰을 하였고, 방사면역측정 법을 이용하여 난소마쇄현탁액내 프로게스테론, 테스토스테론 및 에스트라디올을 정량하였다. 방사선조사로 유강소난포 및 무강소난포가 공히 높은 폐쇄율을 보였고, 난포내 프로게스테론의 농도가 증가되었으며 테스토스테론 및 에스트라디올의 농도는 감소되었다. 즉, 방사선을 조사함으로써 난소내 난포세포인 협막세포에 영향이 미친 결과 $3{\beta}-HSD(3{\beta}-hydroxysteroid\;dehydrogenase) $ 및 isomerase의 활성이 억제 혹은 둔화되어 테스토스테론 및 에스트라디올의 합성이 저하되었고 그 결과 난포의 폐쇄가 가속화되었다고 사료된다.