• Reproductive and Developmental Biology
• /
• v.35 no.3
• /
• pp.355-361
• /
• 2011

Ski protein is implicated in proliferation/differentiation in a variety of cells. We had previously reported that Ski protein is present in granulosa cells of atretic follicles, but not in preovulatory follicles, suggesting that Ski has a role in apoptosis of granulosa 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 the present study was to locate Ski protein in the rat ovary during luteinization to predict the possible role of Ski. 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 gonadotropin to immature female rat, and luteinization was induced by human chorionic gonadotropin 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). Though Ski protein is absent in granulosa cells of preovulatory follicle, its mRNA (c-ski) was expressed and the level was unchanged even after LH surge. Taken together, these results demonstrated that Ski protein expression is induced in granulosa cells upon luteinization, and suggested that its expression is regulated post-transcriptionally.

• Reproductive and Developmental Biology
• /
• v.35 no.3
• /
• pp.341-348
• /
• 2011

Sloan-Kettering virus gene product of a cellular protooncogene c-Ski is an unique nuclear pro-oncoprotein 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. The aim of the present study was to locate Ski protein in rat ovaries in order to predict the possible involvement of Ski in follicular development and atresia. First, expression of c-Ski mRNA in the ovaries of adult female rats was confirmed by RT-PCR. Then, ovaries obtained on the day of estrus were subjected to immunohistochemical analysis for Ski and proliferating cell nuclear antigen (PCNA) in combination with terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). Ski was expressed in granulosa cells that were positive for TUNEL, but negative for PCNA, regardless of the shape and size of follicles. Expression of Ski in TUNEL-positive granulosa cells, but not in PCNA-positive granulosa cells, was also verified in immature hypophysectomized rats having a single generation of developing and atretic follicles by treatment with equine chorionic gonadotropin (eCG). These results indicate that Ski is profoundly expressed in the granulosa cells of atretic follicles, but not in growing follicles, and suggest that Ski plays a role in apoptosis of granulosa cells during follicular atresia.

• Reproductive and Developmental Biology
• /
• v.32 no.2
• /
• pp.135-140
• /
• 2008

Recent studies on nuclear transfer and induced pluripotent stem cells have demonstrated that differentiated somatic cells can be returned to the undifferentiated state by reversing their developmental process. These epigenetically reprogrammed somatic cells may again be differentiated into various cell types, and used for cell replacement therapies through autologous transplantation to treat many degenerative diseases. To date, however, reprogramming of somatic cells into undifferentiated cells has been extremely inefficient. Hence, reliable markers to identify the event of reprogramming would assist effective selection of reprogrammed cells. In this study, a transgene construct encoding enhanced green fluorescent protein (EGFP) under the regulation of human Oct4 promoter was developed as a reporter for the reprogramming of somatic cells. Microinjection of the transgene construct into pronuclei of fertilized mouse eggs resulted in the emission of green fluorescence, suggesting that the undifferentiated cytoplasmic environment provided by fertilized eggs induces the expression of EGFP. Next, the transgene construct was introduced into human embryonic fibroblasts, and the nuclei from these cells were transferred into enucleated porcine oocytes. Along with their in vitro development, nuclear transfer embryos emitted green fluorescence, suggesting the reprogramming of donor nuclei in nuclear transfer embryos. The results of the present study demonstrate that expression of the transgene under the regulation of human Oct4 promoter coincides with epigenetic reprogramming, and may be used as a convenient marker that non-invasively reflects reprogramming of somatic cells.

• The Korean Journal of Physiology and Pharmacology
• /
• v.14 no.5
• /
• pp.265-272
• /
• 2010

Follicular cystic ovary (FCO) is one of the most frequently diagnosed ovarian diseases and is a major cause of reproductive failure in mammalian species. However, the mechanism by which FCO is induced remains unclear. Genetic alterations which affect the functioning of many kinds of cells and/or tissues could be present in cystic ovaries. In this study, we performed a comparison analysis of gene expression in order to identify new molecules useful in discrimination of bovine FCO with follicular cystic follicles (FCFs). Normal follicles and FCFs were classified based on their sizes (5 to 10 mm and $\geq25mm$). These follicles had granulosa cell layer and theca interna and the hormone $17{\beta}$-estradiol ($E_2$)/ progesterone ($P_4$) ratio in follicles was greater than one. Perifollicular regions including follicles were used for the preparation of RNA or protein. Differentially expressed genes (DEG) that showed greater than a 2-fold change in expression were screened by the annealing control primer (ACP)-based PCR method using $GeneFishing^{TM}$ DEG kits in bovine normal follicles and FCFs. We identified two DEGs in the FCFs: ribosomal protein L15 (RPL15) and microtubule-associated protein 1B (MAP1B) based on BLAST searches of the NCBI GenBank. Consistent with the ACP analysis, semi-quantitative PCR data and Western blot analyses revealed an up-regulation of RPL15 and a down-regulation of MAP1B in FCFs. These results suggest that RPL15 and MAP1B may be involved in the regulation of pathological processes in bovine FCOs and may help to establish a bovine gene data-base for the discrimination of FCOs from normal ovaries.

• Development and Reproduction
• /
• v.17 no.3
• /
• pp.269-274
• /
• 2013

This study was performed to evaluate the effects of fibroblast co-culture on in vitro maturation (IVM) of prepubertal mouse preantral follicles. The intact preantral follicles were obtained from the ovaries of 12-14 day old mice and these were cultured individually in ${\alpha}$-minimal essential medium (${\alpha}$-MEM) supplemented with 5% fetal bovine serum (FBS), $100mIU/m{\ell}$ recombinant follicle stimulating hormone (rFSH), 1% insulin-transferrin-selenium, $100{\mu}g/ml$ penicillin and $50{\mu}g/m{\ell}$ streptomycin as base medium for 12 days. A total of 200 follicles were cultured in base medium co-cultured with mouse embryonic fibroblast (MEF) (MEF group) (n=100) or only base medium as control group (n=100). Survival rate of follicles on day 12 of culture were significantly higher in the MEF group of 90.0%, compared with 77.0% of the control group (p=0.021). Follicle diameters on day 6 and 8 of the culture period were significantly larger in the MEF group than those in the control group (p=0.021, p=0.007, respectively). Estradiol levels in culture media on day 4, 6, 8, 10 and 12 of the culture period were significantly higher in the MEF group (p=0.043, p=0.021, p=0.006, p<0.001 and p=0.008, retrospectively). Our data suggest that MEF cell co-culture on IVM of mouse preantral follicle increases survival rate and promotes follicular growth and steroid production.

• Clinical and Experimental Reproductive Medicine
• /
• v.47 no.3
• /
• pp.194-206
• /
• 2020

Objective: The aim of this study was to investigate microRNAs (miRNAs) related to follicle-stimulating hormone (FSH) responsiveness using miRNA microarrays and to identify their target genes to determine the molecular regulatory pathways involved in FSH signaling in KGN cells. Methods: To change the cellular responsiveness to FSH, KGN cells were treated with FSH receptor (FSHR)-specific small interfering RNA (siRNA) followed by FSH. miRNA expression profiles were determined through miRNA microarray analysis. Potential target genes of selected miRNAs were predicted using bioinformatics tools, and their regulatory function was confirmed in KGN cells. Results: We found that six miRNAs (miR-1261, miR-130a-3p, miR-329-3p, miR-185-5p, miR-144-5p and miR-4463) were differentially expressed after FSHR siRNA treatment in KGN cells. Through a bioinformatics analysis, we showed that these miRNAs were predicted to regulate a large number of genes, which we narrowed down to cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and estrogen receptor alpha (ESR1) as the main targets for miR-4463. Functional analysis revealed that miR-4463 is a regulatory factor for aromatase expression and function in KGN cells. Conclusion: In this study, we identified differentially expressed miRNAs related to FSH responsiveness. In particular, upregulation of miR-4463 expression by FSHR deficiency in human granulosa cells impaired 17β-estradiol synthesis by targeting CYP19A1 and ESR1. Therefore, our data might provide novel candidates for molecular biomarkers for use in research into poor responders.

• Clinical and Experimental Reproductive Medicine
• /
• v.32 no.2
• /
• pp.121-132
• /
• 2005

연구목적: 본 연구에서는 비폐쇄성 무정자증 환자에서 나타나는 정자형성과정의 이상과 고환세포의 세포자연사와의 연관관계 여부를 확인하였다. 또한 SELDI-TOF MS 분석을 통하여 고환 내 단백질 발현 양상을 확인하고, 질환에 따른 효과적인 biomarker 개발 가능성 여부를 확인하였다. 재료 및 방법: RT-PCR 및 면역조직화학법을 사용하여 고환에서의 Fas, FasL, Bcl-2, Bax와 Caspase-3의 발현 양상을 확인하고, in situ DNA 3'-end-labelling 방법으로 고환세포의 세포자연사 양상을 확인하였다. SELDI-TOF MS 분석법에 의한 고환의 병리학적 소견에 따른 단백질 발현 변화는 소수성 칩 ($H_4$)을 사용하여 분자량 10~100 kDa 범위 내에서 분석하였다. 결 과: 정상적인 정자형성과정을 보이는 폐쇄성 무정자증 환자의 고환에 비해 지주세포 증후군 (Sertoli cell only syndrome)과 성숙정지 (maturation arrest)를 보이는 고환 내 생식세포와 지주세포에서 세포자연사가 현저하게 증가한 것을 확인할 수 있었다. 세포자연사 관련인자들의 발현 양상을 확인한 결과, 지주세포 증후군과 성숙정지 환자군에서 Fas와 FasL mRNA의 발현이 증가하였으나, bcl-2, bax와 caspase-3 mRNA 발현의 경우에는 두 질환 모두에서 유의한 차이를 확인할 수 없었다. FasL 단백질 발현의 경우, 세포자연사의 증가가 관찰되었던 지주세포 증후군과 성숙정지를 보이는 환자의 간질세포와 지주세포에서 증가하는 양상을 나타내었다. SELDI-TOF MS 분석 결과에서 폐쇄성 무정자증 환자군에 비해 전체적인 단백질 발현양이 지주세포 증후군과 성숙정지 환자의 고환에서 감소하는 양상을 보였으며, 특히, 16.730 kDa 단백질의 현저한 감소를 확인할 수 있었다. 결 론: 본 연구결과를 통해 비폐쇄성 무정자증 환자에서 나타나는 정자형성과정의 장애는 생식세포의 비정상적인 세포자연사와 연관되어 있으며, 고환 내 Fas와 FasL의 비정상적인 발현이 주된 원인인 것을 확인할 수 있었다. 또한, SELDI-TOF MS 분석법을 통한 단백질 발현 양상의 연구는 무정자증 환자에서의 다양한 병리학적 소견을 쉽게 파악할 수 있는 biomarker 발굴뿐만 아니라 질환의 원인규명을 위한 연구에도 유용하게 이용될 수 있을 것으로 사료된다.

• Korean Journal of Clinical Laboratory Science
• /
• v.44 no.1
• /
• pp.31-37
• /
• 2012

Many mammals in temperate zones are affected by the distinctive changes of the four seasons in these zones. Their reproductive status is active in the summer climate and inactive during severe winter weather. The golden hamster (Mesocricetus auratus) is seasonal breeding animal whose sexual activities are regulated by photoperoidism. The reproduction and metabolism are activated by long summer days (LD) and inhibited by short winter days (SD). After several months of SD, animals become refractory to this inhibitory photoperiod and spontaneously revert to LD-like physiology. The suprachiasmatic nuclei (SCN) house the primary circadian oscillator in mammals. Seasonal changes in the photic input to this structure control many annual physiological rhythms via SCN-regulated pineal melatonin secretion, which provides an internal endocrine signal representing photoperiod. The aim of this study was to assess the variation in the morphology of the testis in relation to the natural photoperiod in male golden hamsters. The hamsters were castrated at different weeks (2, 5, 8, and 15). The cell numbers of tubules with spermatogonia (SG), spermatocyte (SC), spermatids (ST), and spermatozoa (SZ) were recorded in each sample. The results showed that testicular regression of golden hamsters occurred in the SD-treated animals. The present investigation determines that the effects of the photoperiod on the reproduction of male golden hamsters. It was also found that the circadian period increases the rate of reproductive inhibition in animals exposed to inhibitory photoperiods.

• Korean Journal of Veterinary Research
• /
• v.55 no.3
• /
• pp.155-161
• /
• 2015

The number of wild animal species is gradually decreasing due to poaching, hunting and habitat loss. While several endangered animal species have been successfully preserved at the zoo, assisted reproductive technology (ART) must be applied to restore wild animals. In the case of critically endangered animals, somatic cell cloning is considered the most appropriate method of ART. Somatic cell cloning can be beneficial for the reproduction of endangered species with limited female populations. However, gene and cell banks, and understanding of reproductive physiology and optimization of ART for wild animals are urgently required for further activation of artificial reproduction of endangered species, which enlarges its application and maintains biodiversity. Care should also be taken to consider ethical and legal issues associated with somatic cell cloning for conservation of endangered animals.

• Reproductive and Developmental Biology
• /
• v.38 no.1
• /
• pp.1-8
• /
• 2014

Artificial insemination technique has been contributed immensely for production of livestock worldwide as a critical assisted reproductive technique to preserve and propagate excellent genes in domestic animal industry. In the past decade, methods for semen preservation have been improved mostly in liquid preservation method for boar semen and freezing method for bull semen. Among many factors affecting semen quality during preservation, reactive oxygen species, produced by aerobic respiration in sperm for survival and motility, are unfavorable to sperm physiology. In mammalian cell as well as in the sperm, antioxidant system plays a role in degradation of reactive oxygen species. Magnetized water forms smaller stabilizing water clusters, resulting in high absorption and permeability of the cell for water, implicating its application for semen preservation. Therefore, this review focuses on preservation methods of boar and bull semen with respect to improvement of extender and reduction of reactive oxygen species by using magnetized water and supplementation of antioxidants.