• 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.

• Journal of Veterinary Clinics
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• v.24 no.1
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• pp.38-41
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• 2007

A 12-year-old, intact female Yorkshire terrier was presented with progressive abdominal distention. On radiographic and ultrasonographic evaluation, a large mass was detected in right upper abdomen and it had several discrete anechoic follicles and large cysts. The mass was removed by complete ovariohysterectomy and the resected specimen was histopathologically examined. Based on the gross and histopathologic findings, the mass was definitely diagnosed as malignant granulosa cell tumor (GCT). Until now, the dog has been successfully managed more than a year without complications.

• Toxicological Research
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• v.33 no.1
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• pp.49-54
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• 2017

Vitamin D3 is a fat-soluble secosteroid responsible for enhancing intestinal absorption of calcium, iron, and other materials. Vitamin D3 deficiency, therefore, can cause health problems such as metabolic diseases, and bone disorder. Female sex hormones including estrogen and progesterone are biosynthesized mainly in the granulosa cells of ovary. In this study, we isolated granulosa cells from porcine ovary and cultured for the experiments. In order to examine the effect of vitamin D3 on the ovarian granulosa cells, the mRNA and protein levels of genes were analyzed by real-time PCR and Western blot assay. The production of estrogen from the granulosa cells was also measured by the ELISA assay. Genes associated with follicle growth were not significantly altered by vitamin D3. However, it increases expression of genes involved in the estrogen-biosynthesis. Further, estrogen concentrations in porcine granulosa cell-cultured media increased in response to vitamin D3. These results showed that vitamin D3 is a powerful regulator of sex steroid hormone production in porcine granulosa cells, suggesting that vitamin D deficiency may result in inappropriate sexual development of industrial animals and eventually economic loss.

• Animal Bioscience
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• v.34 no.6
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• pp.957-966
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• 2021

Objective: Ovarian follicular development, which dependent on the proliferation and differentiation of granulosa cells (GCs), is a complex biological process in which miRNA plays an important role. Our previous study showed that miR-458b-5p is associated with ovarian follicular development in chicken. The detailed function and molecular mechanism of miR-458b-5p in GCs is unclear. Methods: The luciferase reporter assay was used to verify the targeting relationship between miR-458b-5p and catenin beta-1 (CTNNB1), which is an important transcriptional regulatory factor of the Wnt/β-catenin pathway. The cell counting kit-8 (CCK-8) assay, flow cytometry with propidium iodide (PI) and annexin V-fluorescein isothiocyanate (FITC) labeling were applied to explore the effect of miR-458b-5p on proliferation, cell cycle and apoptosis of chicken GCs. Quantitative real-time polymerase chain reaction and Western blot were used to detect the mRNA and protein expression levels. Results: We demonstrated that the expression of miR-458b-5p and CTNNB1 showed the opposite relationship in GCs and theca cells of hierarchical follicles. The luciferase reporter assay confirmed that CTNNB1 is the direct target of miR-458b-5p. Using CCK-8 assay and flow cytometry with PI and Annexin V-FITC labeling, we observed that transfection with the miR-458b-5p mimics significantly reduced proliferation and has no effects on apoptosis of chicken GCs. In addition, miR-458b-5p decreased the mRNA and protein expression of CD44 molecule and matrix metallopeptidase 7, which are the downstream effectors of CTNNB1 in Wnt/β-Catenin pathway and play functional roles in cell proliferation. Conclusion: Taken together, the data indicate that miR-458b-5p regulates ovarian GCs proliferation through Wnt/β-catenin signaling pathway by targeting CTNNB1, suggesting that miR-458b-5p and its target gene CTNNB1 may potentially play a role in chicken ovarian follicular development.

• Clinical and Experimental Reproductive Medicine
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• v.32 no.3
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• pp.207-216
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• 2005

Objective: To understand the crucial requirement for the normal early folliculogenesis, we evaluated molecular as well as physiological differences during in vitro ovarian culture. Among the important regulators for follicle development, anti-Müllerian hormone (AMH) and FSH Receptor (FSHR) have been known to be expressed in the cuboidal granulosa cells. Meanwhile, it is known that c-kit is germ cell-specific and GDF-9 is also oocyte-specific regulator. To evaluate the functional requirement for the competence of normal follicular development, we investigated the differential mRNA expression of several factors secreted from granulosa cells and oocytes between in vivo and in vitro developed ovaries. Materials and Methods: Ovaries from ICR neonates (the day of birth) were cultured for 4 days (for primordial to primary transition) or 8 days (for secondary follicle formation) in ${\alpha}$-MEM glutamax supplemented with 3 mg/ml BSA without serum or growth factors. The mRNA levels of the several factors were investigated by quantitative real-time PCR analysis. Freshly isolated 0-, 4-, and 8-day-old ovaries were used as control. Results: The mRNA of AMH and FSHR as granulosa cell factors was highly increased according to the ovarian development in both of 4- and 8-day-old control. However, the mRNA expression was not induced in both of 4- and 8-day in vitro cultured ovaries. The mRNA expression of GDF-9 known to regulate follicle growth as an oocyte factor was different between in vivo and in vitro developed ovaries. In addition, the transcript of GDF-9 was expressed in the primordial follicles of mouse ovaries. The mRNA expression of c-kit was not significantly different during the early folliculogenesis in vitro. Conclusion: This is the first report regarding endogenous AMH and FSHR expression during the early folliculogenesis in vitro. In conclusion, it will be very valuable to evaluate cuboidal granulosa cell factors as functional marker(s) for normal early folliculogenesis in vitro.

• Journal of Embryo Transfer
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• v.12 no.3
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• pp.293-300
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• 1997

Supplementation of maturation medium with additional granulosa cells has beneficial effect on in vitro maturation of bovine follicular oocytes and their subsequent cleavage and development in vitro. However, maturation system using granulosa cells have some disadvantages that collection of granulosa cells is cumbersome and metabolic activity of the cells is variable according to ovarian cycle or follicular size. We hypothesized that bovine immsture oocytes matured without granulosa cell coculture can fertilize and develop normally if the medium volume per oocyte is reduced during in vitro maturation. Immature oocytes were matured for 24 hours in a TCM199 containing 10% fetal calf serum, anterior pitultary hormone (0.02 AU /ml Antrinⓡ) and estradiol with or without granulosa cells in vitro. In Group 1, 35 to 40 oocytes were matured in a well of 4-well plastic dish containing 500 $\mu$l of maturation medium and granulosa cells, and 9 to 10 oocytes were matured in a 50-$\mu$l drop of maturation medium without granulosa cells in Group 2. After maturation, oocytes were coincubated with sperm for 30 hours in a modified Tyrode's medium (IVF). Inseminated oocytes were cultured in a microdrop (30 $\mu$l) of a synthetic oviduct fluld medium (SOFM) containing BSA, Minimum Essential Medium essential and non-essential amino acids for 9 days. As a preliminary experiment, we investigated the beneficial effect of granulosa cells during maturation on subsequent cleavage and development using the same type of culturedishes (4-well dish). Granulosa cells could not increase embryo cleavage after fertilization but significantly improved (p<0.05) embryo development to expanding blastocyst (Table1 and 2). In Group 1, 68 and 80% of inseminated oocytes have cleaved at 30 hours and 2 days after IVF, respectively, which is similar (p>0.05) to the result of Group 2 (69% at 30 hours and 78% at 2 days after IVF). The oocytes in Group 2 showed 21 and 11% of developmental rates to expanding and hatching blastocysts, respectively, which was not significantly different (p>0.05) from those (20 and 10%, respectively) of oocytes in Group 1. In conclusion, it has been clarified that a microdrop culture system without granulosa cells for in vitro maturation can support bovine embryonic development to blastocyst in vitro as readily as a granulosa cell coculture system.

• 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.

• Journal of Veterinary Clinics
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• v.26 no.5
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• pp.486-489
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• 2009

An 8-year-old Thoroughbred mare showing anestrus for longer than 6 months with left ovarian mass was referred to Veterinary Medical Teaching Hospital of Seoul National University. For 6 months, the mare had received 2 times hormonal therapies with $PGF_2{\alpha}$ and progesterone. The mare had not responded to these therapies and not shown estrous behavior. On rectal palpation, the left ovary was enlarged and contralateral ovary was small and inactive. Transrectal ultrasonographic examination of the left ovary revealed a mass composed of multiple, irregularly shaped follicles. Granulosa cell tumor (GCT) was suspected according to her history. Ovariectomy was performed through the left flank laparotomy with a standing position in a simple chute located outside of horse farm. The mass was diagnosed as GCT on histopathology. The mare was recovered without any problem and became pregnant next spring.

• Clinical and Experimental Pediatrics
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• v.59 no.sup1
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• pp.107-111
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• 2016

Sex cord tumors with annular tubules are known to originate from the sex cord of embryonic gonads that synthesize Sertoli cells, Leydig cells, granulosa cells, and theca cells of the ovarian stroma, while ovarian small cell carcinoma of the hypercalcemic type is a type of neuroendocrine tumor. Both these tumors are uncommon, potentially malignant neoplasms in children. We report the case of a sex cord tumor with annular tubules in an 11-year-old girl and a case of small cell carcinoma of the hypercalcemic type in a 10-year-old girl. We also discuss the prognosis and management of these tumors.

• Development and Reproduction
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• v.10 no.1
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• pp.1-7
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• 2006

Ovarian follicular atresia in mammals is finely regulated by gonadotropins and sex steroid hormones. It is well known that granulosa cell pyknosis is a common cytological feature of atretic follicles in the ovary. The present study hypothesized that granulosa cell pyknosis during follicular atresia might be related to apoptotic process and associated with caspase-3 activation. Healthy (normal) and atretic follicles were isolated from porcine ovaries based on macro-morphological criteria. Isolated follicles were either processed for histological observation or used for collection of granulosa cells by aspiration. Hoechst 33258 staining of the cells showed a significantly higher number of fragmented nuclei, a typical morphological feature of apoptotic cell, in granulosa cells from atretic follicles than those from healthy follicles. In addition, the rate of cell death was significantly higher in granulosa cells from atretic follicles than healthy follicles, as measured by flow-cytometric cell cycle analysis. In situ detection of apoptotic cells by TUNEL revealed that apoptosis was mostly restricted to granulosa cells in follicles. Theca cells were TUNEL-negative. Finally, it has been shown by caspase-3 activity assay that granulosa cells from atretic follicles retain a higher caspase-3 activity compared to healthy follicles. Taken together, it is suggested that granulosa cell degeneration during folliclar atresia occurs by caspase-3-dependent apoptotic fashion.