• Journal of Life Science
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• v.19 no.12
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• pp.1764-1768
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• 2009

This study was carried out to examine a concentration of steroid hormones and in vitro development of mouse embryos in culture supernatant of bovine granulosa cells (GC). To obtain the culture supernatant, granulosa cells were retrieved from mature follicles (6~15 mm diameter) and immature follicles (2~5 mm diameter) of bovine ovary and were cultured, respectively, in media of Ham's F-10 with 15% FCS for 16 days. Mature and immature granulosa cells formed their monolayers easily and showed similar growth patterns in culturing. There was no morphological difference between mature and immature granulosa cells. High levels of both progesterone and estradiol were detected in the culture supernatant of mature granulosa cells and immature granulsa cells, and the endocrine profiles of the two types of cells were similar. Progesterone secretion of granulosa cells was high in the late stage of culturing and estradiol secretion was high in the early stage of culturing. In vitro development rates of mouse embryos to morula, blastocyst and hatched blastocyst were significantly (p<0.05) higher in culture supernatant of mature granulosa cells (92.7%, 78.1% and 34.5%) and in culture supernatant of immature granulosa cells (96.4%, 78.5% and 26.8%) than in Ham's F-10 (86.7%, 41,7% and 13.3%). However, there was no difference between the culture supernatant of mature granulosa cells and the culture supernatant of immature granulosa cells in the development of embryos.

• Journal of Genetic Medicine
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• v.18 no.1
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• pp.1-7
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• 2021

The oocyte quality is of great importance in infertility as it reflects the follicle developmental potential and further affects the embryo development, clinical pregnancy outcomes. The analysis of gene expression in somatic cells is an important study to better clinical in vitro fertilization (IVF) outcomes in embryo selection reflecting the appropriate communication between the oocyte and somatic cells. Specifically, somatic cell transcriptomic technology can help assess biomarkers of oocyte and embryo ability. The present article aims to overview the basic aspect of folliculogenesis and review studies involving changes in candidate gene expression of cumulus or granulosa cell related to clinical outcomes in human IVF.

• Development and Reproduction
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• v.1 no.1
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• pp.79-89
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• 1997

Recent studies have demonstrated that apoptotic cell death plays an important role in the mechanism underlying follicular atresia and luteolysis. However, the mechanisms responsible for initiating these processes have not been elucidated. In in vitro fertilization (IVF) programs, it is highly possible that continuous and repeated administration of FSH/hMG and GnRH agonists for the usage of ovarian hyperstimulation may induce apoptotic death of granulosa cells leading to atresia in the human ovarian follicles. The present study was performed to investigate whether FSH/hMG and GnRh agonists used for a longer period in controlled ovarian hyperstimulation has any effect on the apoptosis of granulosa-luteal (GL) cells obtained from hyperstimulated ovaries. To examine apoptotic cell death in the GL cells, cells were stained with acridie orange followed by observed in some of GL cells. Similar but distinct staining of apoptotic GL cells was observed when the cells were examined by using in situ TUNEL method. The healthy-looking cells with normal nuclear morphology were not stained, whereas cells with pyknotic nuclei or with apoptotic nuclei were intensively stained. After examining the ultrastructural features of GL cells by TEM, it was confirmed that the majority of cells seemed to have normal nuclei while GL cells undergoing apoptotic cel death were rarely found. The DNA extracted from GL cells showed a typical pattern of fragmentation following DNA electrophoretic analysis. We have confirmed that the apoptosis occurs in granulosa-luteal cells obtained from hyperstimulated ovaries. Technically, in situ apoptosis detection method is simple and reproducible and is well suited to identify the quality of oocytes retrieved from hyperstimulated ovaries.

• Proceedings of the Korean Society of Veterinary Clinics Conference
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• 2008.10a
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• pp.182-182
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• 2008

• Journal of Animal Science and Technology
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• v.46 no.4
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• pp.571-584
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• 2004

It is known widely that granulosa cell apoptosis leads follicular atresia and macrophages exert their effects directly and/or indirectly from the initiation to the completion of follicular atresia by phagocytosis of apoptotic bodies and secretion of various cytokines. However, the site of initiation, propagation routes and the elimination methods of apoptotic bodies, and the time and methods of penetration of macrophages into the follicles are not known completely. Using pig(Yorkshire-breed) ovary, immunohistochemical studies with TUNEL for apoptotic bodies and pig macrophage monoclonal antibody 4E9 for macrophages, and light and transmission electron microscopic observations were performed. In the pig, follicular atresia began with the granulosa cell apoptosis, and the apoptosis of theca intema cells occured at the same time. The apoptosis of granulosa cells initiated randomly within the granulosa cell layer and propagated rapidly into the whole layer. Ultrastructura1ly, apoptotic granulosa cells showed characteristic pyknotic and deformed nucleus and intracytoplasmic vesicles. Apoptotic bodies were eliminated by intact granulosa cells and macrophages. Intact granulosa cells ingested apoptotic bodies transiently, soon after they fell into the apoptosis. Finally, apoptotic bodies and degenerated oocyte were phagocytosed by macrophages. Macrophages entered the ovarian follicle at the time of initiation of granulosa cell apoptosis, and migrated with the progression of apoptosis. By elimination of theca cells, macrophages contributed the completion of follicular atresia These results will provide valuable informations on the study of the interrelation between macrophage and ovarian follicular atresia.

• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.171-178
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• 1989

These experiments were carried out to investigate the effect of a co-culture with granulosa cells on in vitro maturation, fertilization and development of bovine follicular oocytes. The bovine ovaries were obtained at a slaughter house and the follicular oocytes were recovered by aspirating the follicular fluid from the visible follicles of diameter 2-6mm. Bovine oocytes were matured in vitro for 24-26 hr and then fertilized in vitro using epididymal spermatozoa capacitated by preincubation for 2-3hr in BO solution containing BSA(5mg/ml) and caffein(25mM). Eight hours after insemination, the oocytes were cultured in a co-culture system with granulosa cells. The rates of maturation of the follicular oocytes cultured in a co-culture system with granulosa cells were 83.1%, the rate of fertilization of the follicular oocytes culture in a co-culture in a co-culture system with granulosa cells were 76.9%, respectively. No significant difference are observed between control and treatment in maturation and fertilization rates. The rates of embryos developed to 2-, 4-, 8-, 16-cell and monula stages after co-cultured with granulosa cells were 65.8, 57.9, 39.5, 34.2 and 34.2%, respectively. The value for 16-and morula stages were significantly higher (P<0.05) than those of the embryos cultured in the basic medium.

• Journal of Embryo Transfer
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• v.9 no.1
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• pp.1-6
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• 1994

This experiment was investigated the effect of presence of granulosa cells from follicles of different size on bovine oocyte maturation, cleavage and development to late stage. The nuclear and cytoplasmic maturation of oocytes in the IVM-IVF system are critical for subsequent embryo development. Granulosa cells when the co-cultured with oocytes may interact with cumulus-oocytes complexes and influence the development competence of the oocytes. Granulosa cells from medium (2~6 mm) and large(>1O mm) size follicles were recovered by aspiration, washed 3 times by centrifugation at 500 x g for 5 min. and used for co-culture at a concentration of 2~3 x 106 cells/mi. The oocytes were matured in vitro (IVM) for 24 hrs. in TCM-199 supplemented with 35 $\mu$g/ml FSH, 10 $\mu$g/ml LH, 1 $\mu$g/ml estradiol-17$\beta$ and granulosa cells at 39$^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro (IVF) by epididymal spermatozoa treated with heparin for 24 hrs., and then the zygotes were co-cultured in vitro (I VC) with bovine oviductal epithelial cells for 7 to 9 days. The assessment of maturation revealed that Grade J oocytes showed significantly(P

• Clinical and Experimental Reproductive Medicine
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• v.25 no.2
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• pp.179-187
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• 1998

In mammal, lots of follicles start simultaneously their growth but only a few oocytes are ovulated in every sexual cycles. Most of matured and grown oocytes are destined to degenerate by atresia. However, the molecular and physiological mechanisms are not elucidated yet. The present study was designed to establish an induction method of follicular atresia with ketamine or pentobarbitone and evaluate the effect of these anesthetics on oocyte maturation and granulosa cell apoptosis of the mouse ovarian follicle. The percentages of degenerated oocyte and apoptotic granulosa cell in ketamine treated groups were significantly higher than that in controls (58.9% vs 33.5%, p<0.01, degeneration; 44.9% vs 26.6%, p<0.01, apotosis). Futhermore, it was revealed that the concentrations of progesterone in both groups were markedly higher than that in control. In cunclusion, it is considered that ketamine induce an atresia as pentobarbitone, and may be useful for inducing follicular atresia.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.3
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• pp.350-355
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• 2009

Arachidonic acid (AA) is a polyunsaturated fatty acid that is a normal constituent of membrane lipids in animal cells. In addition to its role as a precursor of prostaglandins, AA itself may play an important role in the regulation of cell function. The effect of AA on functions of granulosa cells was investigated in pre-hierarchical small yellow follicles of laying hens. Immuno-cytochemical staining showed that AA ($10^{-7}-10^{-5}$ M) increased the expression of the extracellular matrix glycoprotein laminin, gap junction connexin 43 and protein kinase C (PKC). Therefore, mediated by the PKC signal pathway, AA may regulate the intercellular communication of granulosa cells and follicular development by increasing the expression of laminin and connexin.

• Membrane and Water Treatment
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• v.10 no.2
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• pp.121-125
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• 2019

Chronic exposure to atrazine (ATR) raises concerns about adverse effects on reproductive functions. We tested our previously validated filtering device, the OBP-based filter, onto a biological model constituted of cultured swine granulosa cells treated for 48 h with media conditioned with 0.1 or $10{\mu}M$ ATR evaluating cell viability and steroidogenesis. The tested atrazine concentrations did not change granulosa cell viability and no filtering effects was observed following treatments with media prepared with differently filtered water. As for steroidogenesis, treatment of water with OBP-based filter containing $10{\mu}M$ atrazine completely suppressed the stimulatory effect of $10{\mu}M$ atrazine on progesterone production as well as the inhibitory effect of $0.1{\mu}M$ ATR on estradiol-$17{\beta}$ production by granulosa cells. Our data demonstrate that the impairment of steroidogenesis induced by ATR is effectively removed after water filtration in the experimental device thus suggesting potential use in biotechnological applications on living cells and/or organisms.