Earticle

There were differences in reproductive physiology between miniature pig and normal pig, suggesting differences in estrus and pregnancy. The importance of apoptosis in the development of ovarian follicles and maturation of oocytes. However little is known about the molecular mechanism behind these characteristics. It is known that proper execution of programmed cell death is crucial for normal follicular development. Especially, the plays a very important role of remodeling corpus luteum for successful fertilization to reproductive effect of mammals. In the present study of this experiment was to understand the programmed cell death in normal pig and miniature pig corpus luteum of estrus day 15. Expression of MAP1LC3A and other autophagy- related genes (ATG13, Beclin-1) was significantly higher in normal pig corpus luteum. On the other hand, the expression of apoptosis-related genes (casp-3, BAX) and BCL-2 gene was significantly higher in miniature pig corpus luteum. Expression of genes related to cell metabolism activity (IGF, PCNA) was significantly higher in the normal pig corpus luteum than in the miniature pig corpus luteum. Gene expression of hormone receptor (FSH, LH) was higher than that of miniature pig corpus luteum. Our result is suggest that the expression of LH and FSH hormone in the normal pig is elevated by the high activity of the cell-metabolizing activity gene, and thus, the autophagy is induced. In the miniature pig, the expression of the cell metabolizing activity gene is low and the progesterone hormone mechanism And BCL-2 gene expression are thought to induce apoptosis. This is normal pig and miniature pig corpus luteum of estrus day 15 were able to confirm that shows a significantly different pattern to each other. While the process of programmed cell death in normal pig corpus luteum majorly depended on the type II pathway (autophagy), miniature pig corpus luteum preferred the type I pathway (apoptosis). These differences may contribute greatly to manipulating the reproduction of pigs.

Luteolysis is a cyclical regression of the corpus luteum in many non-primate mammalian species. Prostaglandin F2α (PGF2α) from the uterus and ovary induces functional and structural luteolysis in bovine. The action of PGF2α is mediated by PGF2α receptor located on the luteal steroidogenic and endothelial cell membranes. PGF2α plays an important role in regulating nitric oxide production in endothelial cells of the bovine corpus luteum. Nitric oxide production and nitric oxide synthase activity are stimulated and induced by PGF2α in luteal endothelial cells. Moreover, the reactive oxygen species inhibits progesterone secretion in bovine luteal cells and induces apoptosis. Thus, the interaction between PGF2α and reactive oxygen species provides important aspects in physiology of the corpus luteum forfunctional and structural luteolysis.

The aim of this study was to evaluate the changes of protein patterns in granulosa cells and corpus luteum in ovaries during the estrus cycle in cows. The estrus cycle was devided into five steps of follicular, ovulatory, early-luteal, mid-luteal and late-luteal phases. In results, 61 spots of total 85 spots were repeated on follicular phase and 51 spots of total 114 spots were repeated on ovulatory phase. The 40 spots of total 129 spots were repeated on early-luteal phase and 49 spots of total 104 spots were repeated on mid-luteal phase. Also 41 spots of total 60 spots were repeated on late-luteal phase. On the other hands, the 16 spots were indicated difference in follicular phase and ovulation phase had a difference 10 spots. It was showed difference No. 103 spot in ovulation phase, No. 135 spot in early-luteal phase and No. 175 and 176 spots in mid-luteal phase. Also, the 11 spots were expressed specifically in mid-luteal phase and No. 178 and 179 spots were difference of expression in late-luteal phase. We confirmed that there were 7 spots for ovulation, 4 spots for luteinization and 2 spots for luteolysis. Spot No. 89～93 in ovulation phase were transferrin, and spot No.94～98 were HSP60. Spot No. 103 was Dusty PK, spot No. 135 was OGDC- E2, and spot No. 175 and 176 were Rab GDI beta from luteinization. Spot No. 178 and 179 in luteolysis were vimentin. This results suggest that will be help to basic data about infertility.

난소에서는 내분비계 호르몬의 작용에 의해 생리학적 현상이 일어나며 황체가 생성하게 된다. 이 과정에서 생 식호르몬인 에스트로겐은 난포와 황체에서 분비되어 황 체의 생성과 퇴화의 작용에 관여하게 된다. 에스트로겐은 에스트로겐 수용체–α와 –β의 결합에 의해서 생식호르 몬의 합성을 조절한다. 난소에서는 에스트라디올–17β 가 주로 합성되며 황체의 생리작용과 혈관신생과정의 신호 전달경로 기전에 관여하게 된다. 또한, 에스트로겐은 프 로게스테론의 합성을 조절하는데, 이 작용은 StAR에 의 해 이동된 콜레스테롤이 미토콘드리아 안에서 P450scc가 프레그네놀론으로 전환시키고, 활면소포체로 이동하여 3 β–HSD에 의해 프로게스테론으로 전환된다. 그리고, 에 스트로겐 수용체–α는 프로게스테론 수용체의 프로모터 부위에 결합되어 활성을 촉진하기도 한다. 또한, 에스트 로겐은 뉴런에서 분비되는 키스펩틴에 의해서 분비가 자 극되기도 한다. 이는 고나도트로핀 방출 호르몬의 분비를 자극하여 난포자극호르몬과 황체형성호르몬의 활성을 촉 진한다. 그리고, 에스트로겐과 키스펩틴의 상호작용은 난 포의 발달과 배란, 황체의 생리현상에 직접적으로 관여할 가능성이 있다. 게다가, 황체의 생성되기 위한 환경조건 에는 혈관신생과정과 밀접한 관계가 있다. 혈관신생과정 에서 중요한 역할을 하는 주요인자는 VEGF이며 혈관세 포의 생존, 증식, 이동에 관여하는 신호경로기전의 대표 적인 물질이다. 특히, VEGF–A는 혈관세포의 생리작용에 관여하는 핵심적인 인자이다. VEGF는 수용체 VEGFR–2 와 결합하여 PI3K, Akt, MAPK, ERK 등의 신호전달경로 에 관여한다. 또한, VEGF는 저산소 유도 인자인 HIF–1α 와 결합하여 VEGF의 분비를 자극한다. 그리고, 에스트로 겐은 HIF–1α의 활성을 촉진시키며, Akt의 활성과 mTOR의 작용이 VEGF를 자극하게 된다. 따라서, 황체에 서의 에스트로겐의 역할은 난소와 황체의 생리현상에 깊 이 관여하며 내분비계 호르몬의 합성과 분비를 조절하는 중요한 생식호르몬이다 할 수 있다.

The physiological functions of the ovary and development of the corpus luteum occur through the activation of endocrine hormones. In this process, estrogen, a reproductive hormone, is secreted in the ovarian follicle and corpus luteum and affects corpus luteum formation and regression. Estrogen controls the synthesis of reproductive hormones by binding to estrogen receptor–α and –β. Estradiol–17β, synthesized in the ovary, regulates the physiological function of the corpus luteum and the angiogenesis signaling pathway. Estrogen controls progesterone synthesis, which is regulated by StAR-transported cholesterol, P450scc-converted pregnenolone in mitochondria, and 3β-HSD-synthesized progesterone in the smooth endoplasmic reticulum. Estrogen secretion is also stimulated by kisspeptin and regulated by gonadotropin-releasing hormone, follicle-stimulating hormone, and luteinizing hormone. Moreover, the formation of the corpus luteum is closely regulated by angiogenesis. VEGF is an important factor in angiogenesis and plays a role in the survival, proliferation, and migration of endothelial cells. Especially, VEGF–A is a key factor in the physiological functions of endothelial cells. VEGF binds VEGFR–2 and affects the signaling pathways of PI3K, Akt, MAPK, and ERK. Also, VEGF binds to HIF–1α, inducing VEGF secretion. Estrogen promotes the activation of HIF–1α, while the activation of mTOR and Akt stimulates VEGF secretion. Therefore, estrogen is a major reproductive hormone in physiological function and the synthesis and secretion of endocrine hormones in the ovary and corpus luteum.

Progesterone is a key steroid hormone secreted by the ovarian corpus luteum and plays a critical role in regulating the reproductive cycle and maintaining pregnancy in animals. Following ovulation, progesterone prepares the endometrium for embryo implantation by promoting glandular secretion and inhibiting uterine smooth muscle contractions, thereby creating a stable environment for gestation. It also exerts negative feedback on the hypothalamic-pituitary-ovarian axis, suppressing the secretion of follicle-stimulating hormone and luteinizing hormone, and preventing the maturation of additional follicles within the same reproductive cycle. In veterinary reproductive management, progesterone is widely applied through estrus synchronization and pregnancy diagnosis protocols. Hormonal control programs such as controlled internal drug release, Altrenogest, and Ovsynch utilize progesterone or its analogs to improve breeding efficiency, particularly in livestock. Thus, ovarian secretion of progesterone is not only essential for physiological reproduction but also serves as a central mechanism in modern reproductive biotechnology.

황체는 배란 후 난포 내에 발생하는 일시적인 내분비 기관으로, estrous cycle에 따라 형성과 퇴행을 반복한다. 황체의 발생과정에는 새로운 혈관생성이 수반되는데, 이러한 현 상을 혈관신생(angiogenesis)이라고 한다. 황체의 발달은 초기, 중기, 후기로 구분하고, 황 체기마다 그 기능은 다양하게 작용한다. 또한, 황체 내 미세환경에 의한 angiogenesis의 과정도 다이내믹하게 변화한다. 따라서, 본 연구는 황체기의 변화에 따른 angiogenesis의 매커니즘을 명확하게 밝혀내기 위해서 실시하였다. 실험을 위한 황체조직 시료는 난소로 부터 회수하였고, 황체의 형태학적 구분에 따라 황체기를 구분하였다. 황체조직의 mRNA 와 protein은 Trizol (RNAiso Plus)과 protein lysis buffer 등을 처리하여 각각 추출하였다. 황체기별(초기, 중기, 후기) 황체조직에서 주요 angiogenesis 인자인 VEGF, Ang1과 그 수용체의 mRNA 및 protein 발현을 분석하였다. 그 결과, VEGF와 Ang1의 mRNA은 황체 초기, 중기에서 강하게 발현하고, 황체후기에서는 감소하는 양상을 보였다. 또한, 그들의 수용체인 VEGFR2와 Tie2의 mRNA도 초기, 중기에서는 강하게 발현하였으나, 후기에서 는 감소하였다. 그러나, 황체초기, 중기에서 두 수용체의 protein은 VEGFR2가 Tie2보다 강하게 발현하였고, 황체후기에서는 모든 수용체의 protein이 발현되지 않았다. 본 결과를 토대로, 황체 내 혈관신생 과정에서 VEGFR2와 Tie2는 중요한 역할을 할 것이다. 따라 서, 추가적인 세포실험을 통하여 황체 내 혈관세포와 그 외세포의 명확한 세포기능을 알 아볼 필요가 있다.

황체는 난소 내 존재하는 일시적인 내분비 조직으로서, 임신과 관련된 성 호르몬을 분 비하며, 발정주기에 따라 형성과 퇴행을 반복한다. 황체의 다이내믹한 생리적 현상을 연 구하기 위해서는 황체세포의 종류와 역할을 알아야 하는데, 일반적으로 황체는 progesterone을 분비하는 luteal steroidogenic cell과 혈관을 형성하는 luteal endothelial cell로 구성 되어 있다. 따라서 본 연구는 황체를 구성하는 세포의 기능을 알아보기 위하여 실시하였 다. 황체 내의 혈액은 0.75% Na4EDTA를 처리하여 제거하였고, DNase I, collagenase A 및 trypsin-EDTA를 처리하여 세포를 분리하였다. 원심분리를 통하여 분리된 세포는 38°C, 5% CO₂조건에서 16시간동안 배양하였다. 16시간 후, 부착된 세포는 luteal steroidogenic cells (LSCs)로 판단하였으며, 군집으로 성장하는 세포는 luteal endothelial cells (LECs)과 luteal steroidogenic cell-like cells (LSC-like cells)로 판단하고 passage를 실시하였다. 분 리된 세포는 PCR을 이용하여 세포의 종류를 검증하였다. 3beta-hydroxysteroid dehydrogenase (3β-HSD), platelet endothelial cell adhesion molecule (PECAM-1), vascular endothelial cardherin (VE-cadherin), epithelial cardherin (E-cadherin), endothelin-1 (ET- 1), endothelial nitric oxide synthase (eNOS) 및 fibroblast growth factor 7 (FGF7) mRNA 발 현 결과, 우리는 황체로부터 LSCs, LECs, LSC-like cells의 세포를 순수하게 분리된 것을 확인하였다. 그리고 LSC-like cells의 유래를 명확하게 하여 황체에서 일어나는 다이내믹 한 생리현상을 이해하는데 중요한 역할을 할 것이다.

The objective of this work was to analyze the concentrations of progesterone (P4) and estrogen (E2) hormones changed during estrus synchronization in dairy heifers. Estrus synchronization was carried out with CIDRⓇ (Controlled Intravaginal Drug Release) devices. Corpus luteum (CL) was classified into three grades based on its size and palpable characteristics. The concentrations of P4 and E2 were measured by enzyme-amplified chemiluminescence. Serum P4 concentration was markedly low at the estrus stage (36 hrs after removal of CIDR) compared to other stages, while E2 concentration was kept high during estrus stage. The serum P4 concentration was highest in the CL classified into gradeⅠ. These results indicate that P4 concentration could be used as a criteria for determining recipients for artificial insemination or embryo transfer in dairy cattle.

The size and location of the corpus luteum and the presence of coexistent follicles are crucial factors in synchronizing recipients and determining the suitability for embryo transfer. However, there has been a recent decline in conception rates after embryo transfer, which is attributed to environmental pollution, uterine inflammation, ovarian cysts, and other factors. Therefore, we conducted experiments to establish a novel criterion for successful embryo transfer assessment. To assess the suitability for embryo transfer one day before transfer, we conducted ultrasound examinations equipped with a vaginal probe to evaluate the corpus luteum and coexistent follicle. We found that instances with corpus luteum and coexistent follicles (diameter: > 10 mm) constituted the majority (69.7%) of cases. When comparing the fertility rates of cases in which the corpus luteum and coexistent follicle (diameter: > 10 mm) were located on the same ovary and cases in which they were not, higher fertility rates were observed when the corpus luteum and coexistent follicle (diameter: > 10 mm) were on different ovaries. Our study revealed a high incidence of corpus luteum and coexistent follicles with a diameter exceeding 10 mm. Therefore, our findings suggest that the co-occurrence of the corpus luteum and a large follicle can serve as a new standard for the evaluation of embryo transfer suitability.

Luteal cells produce progesterone that supports pregnancy. Steroidogenesis requires coordination of the anabolic and catabolic pathways of lipid metabolism. In the present study, the corpus luteum (CL) in early pregnancy established from luteal phase and pregnant phase was analyzed. The first study determined progesterone changes in the bovine CL at day 19 (early maternal recognition period) and day 90 in mid-pregnancy and compared them to the CL from day 12 of the estrous cycle. CL alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF). Comparing CL from luteal phase to those from pregnant phase counterparts, significant changes in expression level were found in 23 proteins. Of these proteins 17 were not expressed in pregnant phase CL but expressed in luteal phase counterpart, whereas, the expression of the other 6 proteins was limited only in pregnant phase CL. Among these proteins, vimentin is considered to be involved in regulation of post-implantation development. In particular, vimentin may be used as marker for CL development during pregnancy because the expression level changed considerably in pregnant phase CL tissue compared with its luteal phase counterpart. Data from 2-DE suggest that protein expression was disorientated in mid pregnancy from luteal phase, but these changes was regulated with progression of pregnancy. These findings demonstrate CL development during mid-pregnancy from luteal phase and suggest that alternations of specific CL protein expression may be involved in maintenance of pregnancy.

Sloan-Kettering virus gene product of a cellular pro-oncogene c-Ski is an unique nuclear pro-onco protein and belongs to the Ski/Sno proto-oncogene family. Ski plays multiple roles in a variety of cell types, it can induce both oncogenic transformation and terminal muscle differentiation when expressed at high levels. Ski protein is implicated in proliferation/differentiation in a variety of cells. The alternative fate of granulosa cells other than apoptosis is to differentiate to luteal cells, however, it is unknown whether Ski is expressed and has a role in granulosa cells undergoing luteinization. Thus, the aim of this study was, by means of immunohistochemical methods, to locate Ski protein in the rat ovaries during ovulation and corpora lutea(CL) formation to predict the possible involvement of Ski in luteinization. In addition, to examine whether the initiation of luteinization with luteinizing hormone(LH) directly regulates expression of Ski in the luteinized granulosa and luteal cells after ovulation by in vivo models. In order to examine the expression pattern of Ski protein along with the progress of luteinization, follicular growth was induced by administration of equine chorionic gonadtropin to immature female rat, and luteinization was induced by human chorionic gonadtropin treatment to mimic luteinizing hormone(LH) surge. While no Ski-positive granulosa cells were present in preovulatory follicle, Ski protein expression was induced in response to LH surge, and was maintained after the formation of corpus luteum(CL). These results indicate that Ski is profoundly expressed in the luteinized granulosa cells and luteal cells of CL during luteinization, and suggest that Ski may play a role in luteinization of granulosa cells.

The aim of this study was to evaluate the changes of protein patterns in granulosa cells and corpus luteum during the estrus cycle in bovine ovary by proteomics ^techniques. Our study was devided into five steps for follicular, ovulatory, early-lteal, midluteal and late-luteal. The protein was extracted from glanulosa cell and corpus luteum proteins by using M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was 700 μg. Immobilized pH gradient (IPG) strip was used 18 cm and 3 11 NL. SDS-PAGE was used 10% acrylamide gel. The protein spots were visualized by Coomassie Brilliant Blue (CBB) staining, analyzed by MALDI mass spectrometry and searched on NCIBlnr. As the result, 61 spots of total 85 spots were repeated on follicular stage and 51 spots of total 114 spots were repeated on ovulatory stage. 40 spots of total 129 were repeated on early-luteal and 49 spots of total 104 spots were repeated on mid-luteal stage. Also 41 spots of total 60 spots were repeated on last-luteal stage. There were differences in the ovulation (follicular∼ovultory stage) in which the spots of follicular stage 19 was only and in ovulation stage was 10 spots. The difference between the luteinization (ovultory∼mid-luteal stage) was the spots counted in each stage. The spots of ovulatory stage was 1, early-luteal stage was 1 and in mid-luteal stage was 2. Eleven spots were found in mid-luteal stage and 2 spots were found in last-luteal stage. In conclusion, we confirmed that there were 7 spots in ovulation, 4 spots in luteinization and 2 spots in luteolysis. Spot No. 89-93 from ovulation were transferrin, and spot No.94 and 95 were HSP60. Spot No. 103 were Dusty PK, spot No. 135 were OGDC-E2, and spot No. 175, 176 were Rab GDI beta from luteinization. Spot No. 178 and 179 from luteolysis were vimentin.