• Applied Microscopy
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• v.37 no.2
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• pp.135-142
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• 2007

This research investigated morphological changes of ovarian follicle according to dose of irradiation when adult mice were exposed to X-rays from 6 MeV LINAC. At day 3 after irradiation of 200 cGy, 400 cGy and 600 cGy X-ray to the hole body of mice, the ovaries collected and stained with TUNEL. The normal follicles and atretic follicles were identified to apoptosis by the staining with TUNEL. In the atretic follicles of the normal ovary, the apoptotic bodies were well appeared and stained brown color. Almost of the follicles following irradiation are stained with TUNEL, but the sensitivity of reaction is weaker than that in irradiation of 400 cGy and 600 cGy X-ray. The granulosa cells of the radiated normal follicle by 400 cGV are shown brown color. In this stage, the nucleus of granulosa cells in the atrectic follicles are condensed and picknotic feature. The size of the radiated follicle by 600 cGy are decreased than the normal follicles. The atropic follicles are filled with apoptotic bodies which change of granulosa cells and theca cells by influence of X-ray. All of cell in the follicles are strongly positive stained with TUNEL by irradiation of 600 cGy.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.99-103
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• 2011

Transducin-like enhancer protein 1(TLE-1) is protein associated with cell proliferation. This study analyzed change of TLE-1 mRNA expression during in vivo and in vitro maturation in porcine oocytes. Oocytes and granulose cells were collected from follicles of <2 mm, 2~6 mm and >6 mm in diameter in slaughtered pig's ovaries. Oocytes collected from follicles of 2~6 mm in diameter were used after in vitro maturation for 0, 10, 20 and 44 h. Cumulus cells and granulose cells were collected after treatment with hyaluronidase. In results, TLE-1 mRNA expression in oocytes collected from follicle >6 mm in diameter is increased, TLE-1 RNA expression in cumulus cells and granulosa cells from follicles <2 mm, 2 mm 6 mm and >6 mm in diameter. However, there is no significant difference. On the other hand, TLE-1 mRNA expression from oocytes cultured for 10 hand 44 h is increased, TLE-1 mRNA in cumulus cells cultured for 10 h is significant increased(p<0.05) than other culture periods. In conclusion, these results show that TLE-1 is expressed in all cell types of oocytes, cumulus cells and granulose cells, and associated with oocyte maturation.

• Journal of Embryo Transfer
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• v.9 no.3
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• pp.261-268
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• 1994

Immatured bovine follicular oocytes added with serum, hormones, granulosa cells and bovine oviduct epithelium cells were fertilized in vitro after in vitro maturation. In vitro maturation and early development capacity were examined and IVF-derived embryos were transferred and to recipients and effects of sperm treatment on in vitro capacitation were investigated. The rate of in vitro maturation was improved when they were co-culutred with granulosa cells in the TCM199 medium added with 10% FCS and hormones. The percentage of acrosome reaction was not differed between sperm treatments and sperm of above 25% under-went AR during 30 min preincubation with caffeine and heparin. The cleavage rate of oocytes in vitro fertilized in TCM199 medium added with 10% FCS and hormones, GC or BOEG higher than that in medium with 10% FCS and GC. But the rate was not significantly different between GC and BOEG The cleavage of rate oocytes cultured in medium containing serum, hormones and BOEG was 80.2% and more embryos were developed to Blastocyst (17.3%). The selected embryos were transferred to 9 recipients by surgical or nonsurgical method but did not result in pregnancy.

• Korean Journal of Animal Reproduction
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• v.22 no.1
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• pp.51-59
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• 1998

This study was undertaken to investigate the effects of ITS and EGF on embryonic development of in vitro matured and fertilized bovine oocytes in culture medium su, pp.ementing with or without calf serum. When fertilized oocytes were cultured in TCM-199 containing 0, 0.1, 0.5 and 1.0\ulcorner/ml ITS with 5% calf serum, the rates of development to blastocyst stage and the cell number of blastocysts were not significantly different among all treatments. And also monolayer of cumulus/granulosa cells prepared in containing calf serum and ITS were no beneficial effects of embryonic development. On the other hand, when EGF was su, pp.imented to TCM-199 containing calf serum or calf serum free, embryonic development rates(24.0 2.8% to 29.2 1.7% or 8% to 9%) and cell number of blastocysts(p<0.05) were significantly increased compared with EGF-free(22.1 2.1 or 1.0%, p<0.05). But when fertilized oocytes were cultured with cumulus/granurosa cells in TCM-199 containing EGF and calf serum, the rate of embryos development to the blastocyst stage and cell number of blastocysts were not significantly different compared with EGF-free and any concentrations. These results showed that ITS and EGF was not improved the development of bovine embryo in vitro matured and in vitro fertilized with calf serum and/or monolayer of cumulus/granulosa cells.

• Korean Journal of Veterinary Research
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• v.43 no.4
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• pp.555-561
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• 2003

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.

• Development and Reproduction
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• v.13 no.4
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• pp.353-362
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• 2009

GnRH and its receptor are known to express locally in the ovary and to regulate the ovarian function by affecting on granulosa and lutein cells. It has been reported that GnRH directly causes apoptosis in the granulosa and lutein cells of the ovary. However, whether the apoptosis of the cells by GnRH is recovered by FSH as an anti-apoptotic factor is not yet known. In this study, we evaluated the apoptosis and the production of progesterone $(P_4)$ and estradiol $(E_2)$ after treatment with 5, 50, and 100 ng/$m\ell$ GnRH and 1 IU/ml FSH in the granulosa-lutein cells that are obtained during oocyte-retrieval for IVF-ET. Results of DNA fragment analysis and TUNEL assay demonstrated that DNA fragmentation and the rate of apoptotic cells were increased in a dose-dependent manner showing a significant increase in the cells treated with 100 ng/$m\ell$ GnRH. In addition, we found that FSH suppresses the apoptosis of the cells induced by GnRH. In the results of chemiluminescence assay for $P_4$ and $E_2$, $P_4$ production was decreased by GnRH treatment, whereas $E_2$ production was not changed. We also demonstrated that FSH inhibits the suppressive effect of GnRH on $P_4$ production as the result of apoptosis. The present results suggest that GnRH agonist using in ovarian hyperstimulation protocol might induce the dysfunction of the ovary, but its function could be recovered by FSH. These results also will be expected to use as the basic data to elucidate the physiological role of GnRH and to develop new ovarian hyperstimulation protocols for IVF-ET.

• Korean Journal of Veterinary Research
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• v.33 no.4
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• pp.757-762
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• 1993

The ovaries of Korean Native cows or heifers were obtained from an abattoir and kept on 20 to $25^{\circ}C$ and transported to laboratory within 2 hrs. The follicular oocystes were collected from 2~6mm follicles in diameter and classified into 3 grades by the morphology of cumulus cells attached. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with $23{\mu}g/ml$ FSH, $10{\mu}g/ml$ LH, $1{\mu}g/ml$ estradio-17 ${\beta}$ and granulosa cells at $39^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by incubation for 12 hrs. of epididymal spermatozoa pretreated with heparin, and then the zygotes were co-cultured in vitro(IVC) with oviductal epithelial cells for 7 to 9 days. Assessment of maturation revealed that 93.0%(147/158) of grade I oocytes had expanded of cumulus cells, which was higher(p<0.05) than the 79.4%(85/107) of grade II oocytes. Compared to epididymal sperm(32.9%), the insemination with frozen and thawed sperm resulted in slightly lower(20.5%), but not significant, development to morulae and blastocysts from grade I oocytes. Co-culture of bovine IVF embryos with oviductal epithelial cells improved the development to transferable embryos significantly(38.1%), compared to co-culture with granulosa cells(20.0%). When VF bovine embryos were vitrified at blastocyst, the post-thaw survival rate was obtained higher resulf for 1 min. equilibration time(82.6%) or 2 min.(73.9%) than 3 min.(18.2%) in EFS solution.

• Clinical and Experimental Reproductive Medicine
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• v.12 no.1
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• pp.83-98
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• 1985

In order to study the mechanism of follicular atresia, the follicles of the porcine ovary were isolated according to the presence or absence of the corpus luteum and their size, and then classified to the normal? or atretic?follicle on the morphological observation such as the transparency, the vascularization of follicle, the nuclear phase of oocyte, and the homogeneity of the granulosa cell layer. The viability of granulosa cells was examined. The concentrations of progesterone ($P_4$), testosterone (T), and estradiol-17 beta ($E_2$) in each follicular fluid were estimated by the radioimmunoassay. The viability of granulosa cells in the atretic follicle was much lower than that of the normal one. The concentration of each steroid hormone increased as the follicular size was increased, was not different in quantity between the normal- and the atretic follicle of which diameter was below 3mm, and were much higher in the atretic follicle than those in the normal one of which diameter was above 7mm. The ratio of the concentration of E2 to T in the large atretic follicle valued higher than that in the normal one, but smaller in the small and medium atretic follicle than that in the normal one. The present study suggests that the mechanism of atresia of the large follicle may be different from that of the small and the medium follicle and that the amount of steroid hormones regarded as the one of the criteria for the atretic follicles.

• Asian-Australasian Journal of Animal Sciences
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• v.24 no.9
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• pp.1204-1210
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• 2011

Objective of this study was to examine the effect of sodium nitroprusside (SNP), a nitric oxide (NO) donor on steroid synthesis, growth and apoptosis of buffalo granulosa cells (GCs) in vitro. Follicular fluid of antral follicles (3-5 mm diameter) was aspirated and GCs were cultured in 0 (control), $10^{-3}$, $10^{-5}$, $10^{-7}$, $10^{-9}\;M$ of SNP for 48 h. To evaluate whether this effect was reversible, GCs were cultured in presence of $10^{-5}\;M$ SNP+1.0 mM $N^{\omega}$-nitro-L-arginine methyl ester (L-NAME) a NO synthase (NOS) inhibitor or hemoglobin (Hb, $1.0{\mu}g$) as NO scavenger. Nitrate/nitrite concentration was evaluated by Griess method, progesterone and estradiol concentrations by RIA and apoptosis by TUNEL assay. SNP ($10^{-3}$, $10^{-5}$, $10^{-7}\;M$) significantly (p<0.05) inhibited estradiol and progesterone synthesis, growth, disorganized GCs aggregates and induced apoptosis in a dose dependent manner. However, $10^{-9}\;M$ SNP induced the progesterone synthesis and stimulated GCs to develop into a uniform monolayer. Combination of SNP $10^{-5}$ M+L-NAME strengthened the inhibitory effect while, SNP+Hb together reversed these inhibitory effects. In conclusion, SNP at greater concentrations ($10^{-3}$, $10^{-5}$ and $10^{-7}\;M$) has a cytotoxic effect and it may lead to cell death whereas, at a lower concentration ($10^{-9}\;M$) induced progesterone synthesis and growth of GCs. These findings have important implications that NOS derived NO are involved at physiological level during growth and development of buffalo GCs which regulates the steroidogenesis, growth and apoptosis.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2005.07a
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• pp.38-38
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• 2005