• Korean Journal of Animal Reproduction
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• v.17 no.4
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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.

• Applied Microscopy
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• v.44 no.1
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• pp.15-20
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• 2014

Cytokeratin 19 (CK19) expressed in epidermis of skin, bulge region of hair follicle, outermost layer of outer root sheath and proximal and distal to bulge. Vimentin is a fibrous protein that localized in cytoplasm of fibroblast and forms cytoskeleton to maintain shape of cell and nucleus. In this study, CK19 and vimentin in skin were confirmed with light, fluorescence and transmission electron microscope. As a result, CK19 was localized epidermis, hair follicles, outer root sheath and nucleus of Merkel's cell. However, vimentin was localized some epidermis, dermis, hypodermis and nucleus of Merkel's cell. The role of CK19 is self-renewal and homeostasis in skin. Also, hair follicle regeneration and hair growth is known to be related. It is supposed that required of structural proteins that make up cytoskeleton is increased. Thereby, expression of CK19 is increased. It is considered that vimentin localized in order to stabilize structure of cell and cytoskeleton of fibroblasts. Also, CK19 and vimentin present in nuclei of Merkel's cell, and to act as a fibrous protein that make up end of a nerve fiber present in Merkel's cell and paracrine function of Merkel's cell.

• BMB Reports
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• v.41 no.4
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• pp.322-327
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• 2008

In this study, we compared the gene expression profiles of non-syndromic hyperplastic dental follicle (HDF) fibroblasts and normal dental follicle (NDF) fibroblasts using cDNA micro-arrays, quantitative PCR, and immunohistochemical staining. Microarray analysis showed that several collagens genes were upregulated in the HDF's, including collagen types I, IV, VIII, and XI and TIMP-1, -3, and -4 (fold ratio > 2.0). In contrast, the expression of MMP-1, -3, -10, and -16 together with IL-8 was more than two fold downregulated. The differential expression of the genes encoding alkaline phosphatase, MMP-1, -3, -8, and IL-8 was confirmed by quantitative RT-PCR, while that of 24 HDFs and 18 NDFs was confirmed by immunohistochemical analysis. However, HDFs showed stronger expression of MMP-3 than NDFs (P < 0.001). Collectively, these results indicate that defective regulation of MMPs mediating connective tissue remodeling may be responsible for abnormal tooth eruption.

• Proceedings of the KACD Conference
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• 2003.11a
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• pp.546.2-546
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• 2003

Dental follicle is the mesenchymal tissue which surrounds developing tooth germ. During tooth root development, periodontal components such as cementum, periodontal ligament and alveolar bone are considered to be created by progenitors present in the dental follicle. However, little is known about these progenitors. Previously we observed that cultured bovine dental follicle cells (BDFC) contained putative cementoblast progenitors. To further analyze the biology of these cells, we have attempted to immortalize BDFC by expression of the polycomb group protein Bmi-1 and human telomerase reverse transcriptase (hTERT). The BDFC expressing Bmi-1 and hTERT showed extended life span by 90 population doublings more than normal BDFC, and still contained cells with potential to differentiate into cementoblasts upon implantation into immunodeficiency mice. Among them, we established a clonal cell line designated as BCPb8, which formed cemetum-like mineralized tissue reactive to anti-cementum specific monoclonal antibody, 3G9, and expressed mRNA for bone sialoprotein, osteocalcin, osteopontin and type I collagen upon implantation. Thus with the combination of hTERT and Bmi-1, we succeeded in immortalization of cementoblast progenitor in BDFC without affecting differentiation potential. The BCPb8 progenitor cell line could be a useful tool not only to study cementogenesis but also to develop regeneration therapy for periodontitis.

• Clinical and Experimental Reproductive Medicine
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• v.33 no.1
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• pp.15-23
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• 2006

Objective: Pituitary adenylate cyclase-activating polypeptide (PACAP), a novel hypothalamic neuropeptide, has been suggested to play a role in ovarian folliculogenesis. The present study evaluated the effect of PACAP on the growth of preantral follicles. Methods: Preantral follicles were mechanically isolated from ovaries of 21-day-old rats and cultured in groups for 3 days in serum-free medium in the absence or presence of PACAP-38 ($10^{-6}M$). Results: Treatment with PACAP-38 resulted in an increase in follicle diameter by 75% whereas treatment with follicle stimulating hormone (FSH) increased follicle diameter by 65%. PACAP-38 treatment enhanced the granulosa cell proliferation as measured by thymidine incorporation analysis. Furthermore, the production of progesterone by cultured granulosa cells and GFSHR-17 cell line was stimulated by PACAP-38. Interestingly, PACAP enhanced FSH action on stimulation of SF-1 and aromatase gene expression. Conclusion: The present results demonstrate that PACAP stimulated preantral follicle growth by potentiating proliferation and by stimulating steroidogenesis.

• Reproductive and Developmental Biology
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• v.33 no.3
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• pp.183-187
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• 2009

This study analyzed change of proteins in granulosa cells during the porcine follicuar development by proteomics techniques. Granulosa cells of the follicles, of which the diameter is $2{\sim}4\;mm$ and $6{\sim}10\;mm$, were collected from ovary of slaughtered pig that each follicle of diameter $1{\sim}4\;mm$ and $6{\sim}10\;mm$. We extracted glanulosa cell proteins by M-PER Mammalian Protein Extraction Reagent. Proteins were refined by clean-up kit and quantified by Bradford method until total protein was $200{\mu}l$. Immobilized pH gradient(IPG) strip used 18 cm, $3{\sim}10\;NL$. SDS-PAGE used 10% acrylamide gel. After silver staining, Melanie 7 and naked eye test were used for spot analyzation. Increasing proteins in glanulosa cell of $6{\sim}10\;mm$ follicle were 7 spots. This spots were analyzed by MALDI-TOF MS and searched on NCBInr. In results, 7 spots were similar to zinc/ling finger protein 3 precursor (RING finger protein 203), angiomotin, heat shock 60 kDa protein 1 (chaperonin) isoform 1 (HSP60), similar to transducin-like enhancer protein 1 (TLE 1), SH3 and PX domains 2A (SH3PXD2A). Those proteins were related with transfer between cells. Increase of proteins has an effect on follicular development.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.31-31
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• 2001

This study was to establish in uitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, This study was to establish in vitro culture system of mouse preantral follicles and to obtain higher in vitro development rates and production of live young. Preantral follicles were obtained from 12-day-old FI mouse (C57BL $\times$ CBA) by enzymatical methods. Oocyte-granulosa cell complexes (OGCs) of preantral follicles were loaded on Transwell-COL insert and cultured in $\alpha$MEM supplemented with 5% FBS, 100 mIU/$m\ell$ FSH and 100 mIU/$m\ell$ hMG for IVG. IVM was performed in $\alpha$MEM supplemented 1.5 IU/$m\ell$ hCG for 18 hrs and IVF was carried out in Ml6 medium. Embryos were cultured in modified Ml6 medium supplemented 10% FBS for 4 days. The effect of the OGCs size on the nuclear/cytoplasmic maturation was significantly higher in 120-150 ${\mu}{\textrm}{m}$ (MII: 33.0%, $\geq$2-cell: 36.7%, $\geq$morula: 20.9%) than in 70-110 ${\mu}{\textrm}{m}$ (MII: 12.2%, $\geq$2-cell: 10.2%, $\geq$morula: 4.8%) (p<0.001). In period of the IVG days, the rate of $\geq$2-cell was significantly higher in 10 days(38.2%) than in 12 days (20.0%) (p<0.01). In period of IVF time, 9 hrs ($\geq$2-cell: 31.5%, $\geq$ morula: 14.3%) indicated significantly higher cytoplasmic maturation rate than 4 hrs ($\geq$2-cell: 17.5%, $\geq$morula: 4.8%) and 7 hrs ($\geq$2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.morula: 4.8%) and 7 hrs (2-cell: 20.4%, $\geq$morula: 6.1%) (p<0.01). However, there was no difference in cytoplasmic maturation between co-cultured preantral follicle ( $\geq$morula: 17.4%) and preantral follicle cultured in Ml6 ( $\geq$morula: 17.4%). 22 morula and blastocysts produced in above optimal condition were transferred to uterus of 2 pseudopregnant recipients, 1 recipient was pregnant and then born 1 live young. This result demonstrates that in vitro culture system of preantral follicles can be used efficiently as another method to supply mouse oocyte.

• The Korean Journal of Malacology
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• v.30 no.4
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• pp.333-342
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• 2014

Ultrastructural studies of oogenesis in oocytes, oocyte degeneration associated with the follicle cells in female Coecella chinensis were investigated for clams collected from Namhae, Geongsangnam-do, Korea. In this study, vitellogenesis during oogenesis in the oocytes occured by way of endogenous autosynthesis and exogenous heterosynthesis. Of two processes of vitellogenesis during oogenesis, the process of endogenous autosynthesis involved the combined activity of the Golgi complex, mitochondria and rough endoplasmic reticulum. whereas the process of exogenous heterosynthesis involved endocytotic incorporation of extraovarian precursors at the basal region of the oolema of the early vitellogenic oocytes prior to the formation of the vitelline coat. It is assumed that the follicle cells were involved in the development of previtellogenic and early vitellogenic oocytes and appear to play an integral role in vitellogenesis in the early and late vitellogenic oocytes by endocytosis of yolk precursors, and also they were involved in oocyte degeneration by assimilating products originating from the degenerated oocytes, thus allowed the transfer of york precursors needed for vitellogenesis (through phagocytosis by phagolysosomes after spawning). Follicle cells presumably have a lysosomal system for breakdown products of oocyte degeneration. and for reabsorption of various phagosomes (phagolysosomes) in the cytoplasm for nutrient storage during the period of oocyte degeneration.

• The Korean Journal of Zoology
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• v.31 no.4
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• pp.318-326
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• 1988

여포세포에서 합성된 난황단백질이 난모세포로 이동하는 동안에 난황체가 이 두 종류의 세포사이에 형성되었다가 결국은 난황막으로 전환한다. 단계7까지는 뚜렷하게 보이던 난모세포막과 여포세포막이 소멸되고 그 자리에 전자 밀도가 높은 난황체 물질이 산만하게 축적된다. 난황체는 단계9에서 막성 구조의 일종인 linkage bridge로 둘러싸여 단계11까지는 두께가 5∼7um가 되리 만큼 성숙한다. 단계13에서 난황체는 비로소 난황막으로 전환되는데, 이때 난황막의 두께는 겨우 1 U m에 지나지 않는다. 이러한 두깨의 감소는 난황체 물질이 다량 난모세포 쪽으로 이동한 것으로 생각 되었다. coated vesicle을 포함한 다양한 종류의 과립이 난황체 양쪽에서 관찰되었는데, 난모세포쪽에 출현한 과립은 난황체 물질이 난모세포로 이동되는 구조로 해석되었고 여포 세포쪽에서 관찰된 과립은 주로 난황체의 전자밀도와 동일한 점으로보아 여포세포에서 합성되어 난황체를 형성하는 물질로 이루어진 구조로 해석되었다. As yolk proteins are transported from !he follicle cells into oocvtes, vitelline body forms and changes into a vitelline membrane between the ko celt types during the vitellogenic period. Cell membranes of oocyte and follicle cells surrounding the oocyte disappear at stage 7 and high electron-dense substance of vitelline body simultaneously accumulates sporadically between the cell types. The vitelline body becomes surrounded by linkage bridge, a membranous structure, at stage 9 and greatly increases in thickness to be 5-7 U m thick at stage 11. At stage 13 the vitelline body becomes vitelline membrane, which is now only 1 U m thick, suggesting that much of the substance of the vitelline body has been transported into oocyte. Various types of vesicles including coated vesicles were observed at both sides of th vitelline body. The vesicles occurred at the side of oocyte were interpreted to be structures transported from the vitelline body into oocyte, whereas those found at the side of the follicle cells were thought to be structures made in the follicle cells and fused into the vitelline body.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.12
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• pp.1647-1651
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• 2004

The follicles (1.8 to 7.8 mm in diameter) were recovered from the ovaries in marketed pigs and the number of granulosa cells, the diameter of oocytes obtained from different development stages of the follicles and follicular fluid levels were determined. Correlations between size measurements and cell counts as well as the diameter of antral follicles and oocytes were also investigated. The results indicated that, while expanding in size, follicle numbers decreased with a greater atretic proportion. Granulosa cells increased in numbers continuously and remained unchanged beyond the size of 200 ${mm}^3$ in non-atretic follicles, whereas a sudden drop of granulosa counts was observed in atretic follicles. Follicular fluid, on the other hand, linearly increased its volume with follicle size and differed little between those of non-atretic and atretic follicles. Diameters of oocytes in non-atretic follicles increased to its maximum when follicles expanded to 150 ${mm}^3$ and maintained its size during later follicular expansion. It is concluded that, for in vitro culture, the optimal size of porcine follicle should be between 150 to 180 ${mm}^3$if they are collected from pre-pubertal gilts of marketing size slaughtered in an abattoir.