Expression of pituitary adenylate cyclase activating polypeptide in the adult rat testis by in situ hybridization and immunohistochemistry

고필옥,곽수동,Koh, Phil-ok,Kwak, Soo-dong The Korean Society of Veterinary Science 2001 大韓獸醫學會誌 Vol.41 No.1

Pituitary adenylate cyclase activating polypeptide (PACAP)는 양의 뇌하수체에서 처음 분리 되었고, 뇌하수체 전엽세포의 cAMP의 생성을 자극하며, 흰쥐고환의 정자형성과 steroid 호르몬 형성에 관련한다고 알려져 있다. 이 연구는 성숙한 흰쥐의 고환에서 PACAP mRNA와 그 단백질의 분포를 조사하여 아래와 같은 결론을 얻었다. PACAP mRNA와 그 단백질은 흰쥐의 정세관에서 정자세포의 생성단계에 따라 특이적으로 발현되었다. 이들은 정세관의 발달단계 중 III~VII 기의 정자세포에서 발현되었고, 특히 V 기에서 초기 VII 기의 원형의 정자세포에서 가장 강하게 발현되었다. 이러한 결과는 흰쥐고환의 발달단계에 있는 정자세포에서 합성된 PACAP이 정자형성에 관련된다는 것을 나타내므로, PACAP이 고환의 기능에 중요한 역할을 하는 것을 암시한다. Pituitary adenyl ate cyclase activating polypeptide (PACAP) was originally isolated from the ovine hypothalamus and stimulated cAMP production in anterior pituitary cells. It is known that PACAP stimulates cAMP accumulation and contributes to the spermatogenesis and steroidogenesis in rat testis. The principal aim of this study is to determinate the distribution of PACAP mRNA and protein in adult rat testis. For this study, we used in situ hybridization and immunohistochemistry techniques in adult rat testis. PACAP mRNA was stage specifically expressed in seminiferous tubules. Positive signals of PACAP mRNA were detected in the developing germ cells at stages HI-VII of the epithelial cycle. The strongest signals of PACAP mRNA and protein were detected in round spermatids at stages V to early VII of the cycle. These results demonstrate that PACAP which is synthesised in the developing germ cells contributes to the spermatogenesis in rat testis. Thus, we suggest that PACAP plays a critical role in the function of testis.

흰쥐 난포의 성장과 퇴화에 따른 bcl-2 단백질 발현에 관한 면역조직화학적 연구

고필옥,정성윤,조경제,최완성,곽수동,Koh, Phil-ok,Jeong, Sung-yoon,Cho, Gyeong-jae,Choi, Wan-sung,Kwak, Soo-dong 대한수의학회 1999 大韓獸醫學會誌 Vol.39 No.1

In the mammalian ovary, follicular development and atresia continuously occur during the reproductive cycles. Follicular atresia occurs through granulosa cell apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2 and bcl-xLong are known as inhibitors of apoptosis, whereas bax and bcl-xShort are known as inducer of apoptosis. We thought that bcl-2 protein is associated with follicular development and atresia. But it is not known that the distribution of cells containing bcl-2 protein during follicular development and atresia. Therefore, to examine the distribution of cells with bcl-2 protein during ovarian follicular development and atresia, the immunohistochemistry was used in the rat ovary. Bcl-2 immunoreactivity was localized in the interstitial cells, theca externa cells and granulosa cells around of antrum. All positive signals were observed in the cytoplasm of these cells. Positive signals were strongly observed in the interstitial and theca externa cells of growing antral follicles. While, positive signals were weakly observed in these cells from atretic antral follicles. Positive signals were very weakly observed in the granulosa cells of growing and atretic antral follicles. According to these data, we suggested that bcl-2 proteins which were strongly expressed in the interstitial cells and theca externa cells of growing antral follicles inhibit follicular atresia. And we purposed that bcl-2 proteins regulated follicular development and atresia through the action of bcl-2 gene family.

미성숙과 성숙한 흰쥐 고환에서의 Steroidogenic acute regulatory protein mRNA의 발현

고필옥,곽수동,Koh, Phil-ok,Kwak, Soo-dong 대한수의학회 2000 大韓獸醫學會誌 Vol.40 No.2

The synthesis of steroid hormone starts from cholesterol. Steroidogenic acute regulatory protein (StAR) acutely transfers cholesterol from the outer mitochondrial membrane to the inner in the early step of steroidogenesis. Many kinds of steroid hormone are mainly synthesized in adrenal grand, ovary, and testis. Among the steroid hormone, testosterone is synthesized in Leydig cells of the testis, the production of testosterone significantly increases in adult testis after puberty onset. Therefore, we think that the expression of StAR mRNA in testis will change according to the testicular development. The aim of this study is to determine the distribution of StAR mRNA in immature and adult rat testes and to confirm the functions of StAR in these testes. Thus, in situ hybridization was used in rat testes of the 2, 4, and 10 weeks of age. StAR mRNA was expressed in Leydig cells. Positive signals of StAR mRNA were weakly detected in Leydig cells of the 2 weeks of age. But, StAR mRNA was strongly expressed in Leydig cells of the 4 and 10 weeks of age, where steroidogenesis actively occur. In our results, the pattern of StAR mRNA expression was similar to the pattern of testosterone production in immature and adult rat testes. In conclusion, we can suggest that StAR acts as an important factor to regulate the synthesis of testosterone in Leydig cells of the rat testis.

성 hormone이 rat 자궁 발달에 미치는 영향에 대한 proliferating cell nuclear antigen 항체의 면역조직학적 응용

고필옥,곽수동,Koh, Phil-ok,Kwak, Soo-dong 대한수의학회 1997 大韓獸醫學會誌 Vol.37 No.2

The study was designed to investigate the effects of progesterone and estrogen on the uterus of rats by immunohistochemical methods using Proliferating Cell Nuclear Antigen (PCNA) antibody. Eighteen female rats(Wistar), weighing initially about 300g, were ovariectomized. These rats were divided into four groups, progesterone-treated group, estrogen-treated group, estrogen+progesterone-treated group, and control group, progesterone-treated group was injected with 1mg of progesterone per rat per day for 2 days and estrogen-treated group with $20{\mu}g$ of $17{\beta}-estradiol$ for 3 days and estrogen+progesterone-treated group with $17{\beta}-estrdiol$ for 3 days and then with progesterone for 2 days as above. In gross findings, the uteri were markedly hypertrophied by estrogen treatment but were not affect in size by progesterone treatment. Immunohistochemical investigation was performed on the cell types with higher appearance of PCNA positive reaction cells in four groups. The groups with higher appearance of the stromal cells were ordered as estrogen-treated group, progesterone-treated group, estrogen+progesterone-treated group, and control group. The muscle cells were ordered as progesterone-treated group, estrogen-treated group, estrogen+progesterone-treated group, and control group. Positive reaction cells of the stromal cells were total 4.6 times higher than those of muscle cells. Therefore, the affect of the hypertrophy on the uterus by estrogen was larger than those of progesterone and affect on the uterus by stromal cells were larger than those of muscle cells. The group with more PCNA positive reaction cells of luminal epithelial cells were ordered as control group, progesterone-treated group, estrogen+progesterone-treated group, and estrogen-treated group, and glandular epithelial cells were ordered as estrogen+progesterone-treated group, progesterone-treated group, control group, and estrogen-treated group. It was suggested that estrogen and progesterone did not affect on the proliferating cells of luminal epithelial cells and affection of progesterone on the development of glandular epithelial cell was larger than that of estrogen.

Expression of Bcl-2 and Bax mRNAs During Follicular Development and Atresia in the Rat Ovary

고필옥(Phil Ok Koh),강상수(Sang Soo Kang),최완성(Wan Sung Choi),곽수동(Soo Dong Kwak),조경제(Gyeong Jae Cho) 대한해부학회 1999 Anatomy & Cell Biology Vol.32 No.1

포유류의 난소는 생식주기에 따라 난포의 성장 (follicular development)과 퇴화(follicular atresia)가 주기적으로 일어나며 이중 난포의 퇴화는 programed cell death인 apoptosis에 의해 일어난다. Apoptosis는 bcl-2 gene family에 의해 조절되며 이들 유전자 중 bcl-2는 apoptosis를 억제하는 유전자로, bax는 apoptosis를 유도하는 유전자로 알려져 있다. 본 연구에서는 미성숙한 흰쥐에 PMSG를 투여하여 난포의 성장과 퇴화를 유도한 후 이들 난포에서 bcl-2와 bax mRNA의 발현을 in situ hybridization으로 조사하였다. PMSG를 투여한 후 1일과 2일군에서는 대부분의 난포가 난포강을 형성하였고 과립세포가 균일하게 난자를 둘러싸고 있 었으며 투여후 3일군에서는 성장하는 난포와 난포의 형태가 일그러지고 과립세포층이 얇아지는 퇴화하는 난포들이 관찰되었다. 4일, 5일군에서는 난포의 과립세포층이 얇아지고 응축된 핵이 나타난 퇴화하는 난포들이 관찰되었는데 이들 퇴화하는 난포는 in situ DNA end labeling에 양성반응세포을 보였다. Bcl-2 mRNA에 대한 in situ hybridization에서는 성장난포와 퇴화난포의 기질세포 (interstitial cell), 난포막세포 (theca cell)에서 양성반응이 나타났고 PMSG를 투여한 후 1일, 2일군의 성장난포의 기질세포, 난포막세포에서 강하게 발현되었다. Bax mRNA는 성장난포와 퇴화난포의 기질세포, 난포막세포, 과립막세포 (granulosa cell)에서 양성반응이 나타났고 PMSG를 투여한 후 4일, 5일군의 퇴화난포의 기질세포, 난포막세포와 과립막세포에서 발현되었으므로 bcl-2와 bax의 비율이 세포의 운명을 결정하는데 중요한 역할을 한다는 사실을 확인 할 수 있었다. Bcl-2는 난포의 성장과 퇴화시 기질세포, 난포막세포에서 발현되어 난포의 퇴화를 억제하는 역할을 하며 bax는 퇴화하는 난포의 기질세포, 난포막세포와 과립막세포에서 발현되어 난포의 퇴화를 유도한다고 생각되었다. 따라서, bcl-2와 bax는 기질세포와 난포막세포에서 주로 합성되어 난포의 성장과 퇴화를 조절한다고 할 수 있다. In the mammalian ovary, follicular atresia occurs through apoptosis. Apoptosis is known as the physiological cell death, which is regulated by bcl-2 gene family. In the bcl-2 gene family, bcl-2/bcl-xLong is known as an inhibitor of apoptosis, whereas bax/bcl-xShort is known as an inducer of apoptosis. We thought that these genes are associated with follicular development and atresia. Therefore, the present study used a in situ hybridization to examine the expression of bcl-2 and bax mRNAs during ovarian follicular development and atresia induced by PMSG(15 IU) treatment in the immature rat ovary. Morphological changes were occurred with the manner of five days periodicity after PMSG treatment. One or two days after PMSG treatment, ovaries had growing follicles with antrum and healthy granulosa cells. Three days after treatment, some degenerating follicles which had thinner granulosa cell layers than growing follicles were observed. At day four and five after treatment, degenerating follicles which have pyknotic nuclei and thin distorted granulosa cell layers appeared. These atretic follicles showed positive reaction with in situ DNA end labelling which indicates apoptotic changes. This study showed that bcl-2 mRNA was expressed in the theca and interstitial cells. Growing follicles of one or two days have showed stronger bcl-2 mRNA signals than atretic follicles of four or five days in these cells. Bax mRNA was expressed in the theca cells, interstitial cells, and granulosa cells. Atretic follicles of four or five days showed stronger bax mRNA signals than growing follicles of one or two days in these cells. Expression of bcl-2 mRNA was increased in growing follicles while decreased in atretic follicles. In contrast, expression of bax mRNA was increased in atretic follicles while decreased in growing follicles. Therefore, we confirmed that follicular development and atresia were affected by the change in the ratio of bcl-2 and bax mRNAs. According to these data, we proposed that these two genes are associated with follicular development and atresia.

Neuroprotective effect and mechanism of estradiol in experimental models of focal cerebral ischemia

Phil-Ok Koh(고필옥) 한국실험동물학회 2009 한국실험동물학회 학술발표대회 논문집 Vol.2009 No.2

Ferulic Acid Prevents Middle Cerebral Artery Occlusion-induced Reduction in Thioredoxin Protein

Phil-OK Koh(고필옥),Sang-A Gim,Dong-Ju Park,Fawad-Ali Shah,Young-Min Kim 한국실험동물학회 2014 한국실험동물학회 학술발표대회 논문집 Vol.2014 No.2

한국재래산양 태아와 신생아의 흉골과 늑골 크기에 관한 연구

김종섭,고필옥,조규현,이종환,원청길,Kim, Chong-Sup,Koh, Phil-Ok,Cho, Gyu-Hyen,Lee, Jong-Hwan,Won, Chung-Kil 대한수의학회 2005 大韓獸醫學會誌 Vol.45 No.4

This study was carried out to investigate the skeletal measurement of the sternum and ribs at 60-, 90-, 120-day-old fetuses and neonates of Korean native goats. The total length and width of the ossified part of the sternebrae and ribs were observed at 60-, 90-, 120-day-old fetuses and neonates. The ossification of the ribs from the 1st to the 8th rib was observed in 60-day-old fetuses, but not observed from the 9th to the last rib. In the 90-day-old fetuses, all ribs were observed having ossified parts. In the 60-day-old fetuses, all sternebrae were not observed having ossified parts. All sternebrae were observed having ossified parts in the 90-day-old fetuses. In the 90-day-old fetuses, the length of the ossified parts of each sternebrae was longer than its width. In the 120-day-old fetuses and neonates, the length of the ossified parts of the 1st, 2nd, and 7th sternebrae was longer than its width, but the width of from the 3rd to 6th sternebrae was longer than the length.

임신우에서 발생된 난포의 기능에 대한 면역조직화학적 관찰

곽수동,고필옥,양제훈,원청길,강정부,Kwak, Soo-Dong,Koh, Phil-Ok,Yang, Je-Hoon,Won, Chung-Kil,Kang, Chung-Boo 대한수의학회 2003 大韓獸醫學會誌 Vol.43 No.4

Incidence of estrum or abortions in pregnant cows may be affected by large follicles developed together with corpus luteum in pair ovaries of pregnant cows. But the follicles of pregnant phase were not assessed about histological findings. Determination of the healthy and atretic follicles by presence of proliferative cells or apoptotic cells and histological compositions of follicles would be used as important data on measurements of ovarian functions. This study was focussed mainly to investigate macroscopical, histological and immunohistochemical findings of ovarian follicles of pregnant Korean native cows and dairy cows (Holstein). In immunohistochemical methods, assessments of proliferative cells using PCNA antibody and apoptotic cells using TUNEL methods were performed. The follicles were observed on all 24 pregnant cows (17 Korean native cows and 7 Holstein cows). Follicles of greater than 10 mm in daimeter were developed in 37.5% (9/24 heads) of these pregnant cows. largest follicles from in these cows were $16.0{\times}15.0mm$ in diameter in a Korean native cow(l20 days of gestation), $13.4{\times}10.1mm$ in a Korean native cow(50 days of gestation), $12.9{\times}11.5mm$ in a Holstein cow (120 days of gestation). 40.5% among all follicles having diameter of greater than 1.0 mm in pregnant cows were assessed as atretic follicles and in addition, healthy follicles also showed less in number and smaller in size and thinner in wall layer compared with those of cyclic phase ovaries. In immunohistochemical findings, also proliferative positive cells and apoptotic positive cells on the granulosa cell layers in the healthy follicles of pregnant cows appeared less than on those of cyclic follicles. So these follicles were assessed as weakly active follicles. In large follicles, above positive cells were not nearly appeared but granulosa cell debris were more appeared among the granulosa cells. So these large follicles were assessed as inactvie or atretic follicles. The above findings suggest that small follicles of pregnant phase were weakly active or atretic and large follicles were inactive or atretic.

고라니(Hydropotes inermis) 장골과 천골이 유합된 1례

김종섭,고필옥,조규완,조재현,원청길,Kim, Chong-Sup,Koh, Phil-Ok,Cho, Kyu-Woan,Cho, Jae-Hyeon,Won, Chung-Kil 대한수의학회 2005 大韓獸醫學會誌 Vol.45 No.4

The sacrum fused with ilium in a chinese water deer (Hydropotes inermis) was observed. The sacrum consists of four sacral vertebrae which was fused each other. In the ventral part of the fused pelvis, on the left, there was a 20.19 mm gap at the dorsolateral part of the sacroiliac articular part which is in between the promontory and the cranial border of the first ventral sacral foramen. On the right, there was a 14.34 mm gap at the dorsal part of sacroiliac articulation. In the dorsal view of the fused pelvis, the wing of sacrum and the wing of ilium were fused tightly, so that the boundary of the two bones was not recognizable. The left and right second dorsal sacral foramina and the third dorsal sacral foramen on the left were small. The third dorsal and ventral sacral foramina on the right were not formed.