• Clinical and Experimental Reproductive Medicine
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• v.32 no.3
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• pp.269-277
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• 2005

Objectives: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. Methods: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (${\underline{c}}onnective$ tissue growth factor/${\underline{c}}ysteine$-rich 61/${\underline{n}}ephroblastoma$-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. Results: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. Conclusions: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.267-271
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• 1997

Apoptosis, programmed cell death, is posulated to occur in granulosa cells in ovarian follicular atresia. bcl-2 gene serves as protector from apoptosis and, thus, is associated with increased cell survival. TRPM-2 gene expression has been implicated as a trigger of apoptosis in rat prostate, uterus and mammary gland. Our objective was to determine if bcl-2 and TRPM-2 are expressed in luteinized human GC and, therefore, have regulatory functions for apoptosis in GC. Human GC were obtained via oocyte retrival from the infertile patients stimulated with exogeneous gonadotropins while undergoing IVF. GC were isolated from follicular fluid using Percoll gradient centrifugation. The GC were further purified with anti-CD45 magnetic beads to remove contaminating WBC's. RT-PCR were performed to analyze the mRNA expression of bcl-2 and TRPM-2 in the GC. The PCR primers were designed to amplify a 195 bp fragment of bcl-2 and a 174 bp fragment of TRPM-2. The PCR products were electrophoresed on 4% agarose gel. Three separate experiments indicated that both bcl-2 and TRPM-2 are concurrently expressed in human GC. We cultured granulosa cells with FSH (1 ng/ml) for 1 day to investigate the relative changes of TRPM-2 mRNA level with RNAse protection assay. When we cultured GC with serum free medium for 1 day TRPM-2 mRNA level increased with 1.3 fold, however it was decreased 0.64 fold with FSH. Therefore we conclude that bcl-2 and TRPM-2 are concurrently expressed and that the interaction of their products may be involved in GC apoptosis. And TRPM-2 may be regulated with FSH.

• The Korean Journal of Malacology
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• v.22 no.1 s.35
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• pp.13-22
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• 2006

Gonadosomatic index (GSI), oogenesis and reproductive cycle of Glossaulax didyma were investigated by the cytological histological obserbations and morphometric data. Samples were collected monthly from the intertidal zone of Biin Bay, Seocheon, Korea, for one year. Monthly variations in the GSI showed similar patterns with gonadal development. In the early vitellogenic oocyte, the Golgi complex and mitochondria were invovled in the formation of lipid droplet and yolk precursor. In the late vitellogenic oocytes, the rough endoplasmic reticulum and multivesicular bodies were involved in the formation of profeid yolk granules in the cytoplasm. A mature yolk granule was composed of three components: main body (central core), superficial layer, and the limiting membrane. The spawning season was from early June to late August, and the main spawning occurred between July and August when the seawater temperature was above $19^{\circ}C$. The female reproductive cycle can be classified into five successive stages: early active stage (December to February), late active stage (February to March), ripe stage (April to July), spawning stage (June to August), recovery stage (August to November). Fully mature oocytes were approximately $250-270{\mu}m$ in diameter.

• Animal Bioscience
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• v.34 no.6
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• pp.975-984
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• 2021

Objective: Inbreeding depression of reproduction is a major concern in the conservation of native chicken genetic resources. Here, based on the successful development of strongly inbred (Sinb) and weakly inbred (Winb) Langshan chickens, we aimed to evaluate inbreeding effects on reproductive traits and identify candidate genes involved in inbreeding depression of reproduction in Langshan chickens. Methods: A two-sample t-test was performed to estimate the differences in phenotypic values of reproductive traits between Sinb and Winb chicken groups. Three healthy chickens with reproductive trait values around the group mean values were selected from each of the groups. Differences in ovarian and hypothalamus transcriptomes between the two groups of chickens were analyzed by RNA sequencing (RNA-Seq). Results: The Sinb chicken group showed an obvious inbreeding depression in reproduction, especially for traits of age at the first egg and egg number at 300 days (p<0.01). Furthermore, 68 and 618 differentially expressed genes (DEGs) were obtained in the hypothalamus and ovary between the two chicken groups, respectively. In the hypothalamus, DEGs were mainly enriched in the pathways related to vitamin metabolism, signal transduction and development of the reproductive system, such as the riboflavin metabolism, Wnt signaling pathway, extracellular matrix-receptor interaction and focal adhesion pathways, including stimulated by retinoic acid 6, serpin family F member 1, secreted frizzled related protein 2, Wnt family member 6, and frizzled class receptor 4 genes. In the ovary, DEGs were significantly enriched in pathways associated with basic metabolism, including amino acid metabolism, oxidative phosphorylation, and glycosaminoglycan degradation. A series of key DEGs involved in folate biosynthesis (gamma-glutamyl hydrolase, guanosine triphosphate cyclohydrolase 1), oocyte meiosis and ovarian function (cytoplasmic polyadenylation element binding protein 1, structural maintenance of chromosomes 1B, and speedy/RINGO cell cycle regulator family member A), spermatogenesis and male fertility (prostaglandin D2 synthase 21 kDa), Mov10 RISC complex RNA helicase like 1, and deuterosome assembly protein 1) were identified, and these may play important roles in inbreeding depression in reproduction. Conclusion: The results improve our understanding of the regulatory mechanisms underlying inbreeding depression in chicken reproduction and provide a theoretical basis for the conservation of species resources.

• Journal of the Korea Academia-Industrial cooperation Society
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• v.20 no.9
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• pp.333-340
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• 2019

This study investigated the effects of goat blood serum (gBS), goat follicular fluid (gFF) and gonadotropin (FSH) on the in vitro maturation, fertilization and development of Korean native black goat oocytes. Our results indicate that the gBS combined with FSH treated group showed significantly higher maturation rate than the other groups. Furthermore, blastocyst formation rate was significantly increased in all treated groups, and gBS and gFF combined with FSH treated groups were higher than other groups. However, gene expression levels of BMP15 and GDF9 in COC, both oocyte maturation related genes, remained unaffected after 24 h maturation. The results of the present study indicate that supplementation of the maturation medium with gBS, gFF and FSH is efficacious in improving the in vitro maturation, fertilization and development of Korea native black goat oocytes.

• Journal of Life Science
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• v.29 no.1
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• pp.105-111
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• 2019

In recent years, progress has been made in the search for the development of new anti-cancer agents by employing specific inhibitors of histone deacetylase (HDAC)-6 to block signal transduction pathways in cancer cells. This study examined the effects of tubastatin A (TubA), an HDAC-6 inhibitor, on the growth and development of immature oocytes in murine ovaries using RNA sequencing analysis. The results from a gene set enrichment analysis (GSEA) indicated that the expression of most of the gene sets involved in the cell cycle and control and progression of meiosis decreased in the TubA-treated group as compared with that in germinal vesicle (GV) stage oocytes. In addition, an ingenuity pathway analysis (IPA) suggested that TubA not only caused increased expression of p53 and pRB and decreased expression of CDK4/6 and cyclin D but also caused elevated expression of genes involved in the control of the DNA check point in G2/M stage oocytes. These results suggest that TubA may induce cell cycle arrest and apoptosis through the induction of changes in the expression of genes involved in signal transduction pathways associated with DNA damage and the cell cycle of immature oocytes in the ovary.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.8
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• pp.1112-1121
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• 2019

Objective: Gram-negative bacteria lipopolysaccharide (LPS) has been reported to be associated with uterine impairment, embryonic resorption, ovarian dysfunction, and follicle retardation. Here, we aimed to investigate the toxic effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Methods: First, we developed an in vitro model to study the response of bovine cumulusoocyte complexes (COCs) to LPS stress. After incubating germinal vesicle COCs in $10{\mu}g/mL$ of LPS, we analyzed the following three aspects: the expression levels of the LPS receptor toll-like receptor 4 (TLR4) in COCs, activities of intracellular signaling protein p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-kappa B (NF-${\kappa}B$); and the concentrations of interleukin (IL)-$1{\beta}$, tumor necrosis factor (TNF)-${\alpha}$, and IL-6. Furthermore, we determined the effects of LPS on the maturation ability and parthenogenetic developmental competence of bovine oocytes. Results: The results revealed that LPS treatment significantly elevated TLR4 mRNA and protein expression levels in COCs. Exposure of COCs to LPS also resulted in a marked increase in activity of the intracellular signaling protein p-p38 MAPK and NF-${\kappa}B$. Furthermore, oocytes cultured in maturation medium containing LPS had significantly higher concentrations of the proinflammatory cytokines IL-$1{\beta}$, TNF-${\alpha}$, and IL-6. LPS exposure significantly decreased the first polar body extrusion rate. The cytoplasmic maturation, characterized by polar body extrusion and distribution of peripheral cortical granules, was significantly impaired in LPS-treated oocytes. Moreover, LPS exposure significantly increased intracellular reactive oxygen species levels and the relative mRNA abundance of the antioxidants thioredoxin (Trx), Trx2, and peroxiredoxin 1 in oocytes. Moreover, the early apoptotic rate and the release of cytochrome C were significantly increased in response to LPS. The cleavage, morula, and blastocyst formation rates were significantly lower in parthenogenetically activated oocytes exposed to LPS, while the incidence of apoptotic nuclei in blastocysts was significantly increased. Conclusion: Together, these results provide an underlying mechanism by which LPS impairs maturation potential in bovine oocytes.

• Animal Bioscience
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• v.34 no.4
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• pp.546-557
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• 2021

Objective: If fertilization does not occur within a specific period, the quality of unfertilized oocytes in the oviduct (in vivo aging) or in culture (in vitro aging) will deteriorate over time. Icariin (ICA), found in all species of Epimedium herbs, has strong antioxidant activity, and is thought to exert anti-aging effects in vitro. We asked whether ICA protects oocytes against age-related changes in vitro. Methods: We analyzed the reactive oxygen species (ROS) levels and expression of antioxidant, maternal, and estrogen receptor genes, and along with spindle morphology, and the developmental competence and quality of embryos in the presence and absence of ICA. Results: Treatment with 5 μM ICA (ICA-5) led to a significant reduction in ROS activity, but increased mRNA expression of glutathione and antioxidant genes (superoxide dismutase 1 [SOD1], SOD2, peroxiredoxin 5, and nuclear factor erythroid 2-like 2), during aging in vitro. In addition, ICA-5 prevented defects in spindle formation and chromosomal alignment, and increased mRNA expression of cytoplasmic maturation factor genes (bone morphogenetic protein 15, cyclin B1, MOS proto-oncogene, serine/threonine kinase, and growth differentiation factor-9). It also prevented apoptosis, increased mRNA expression of antiapoptotic genes (BCL2-like 1 and baculoviral IAP repeat-containing 5), and reduced mRNA expression of pro-apoptotic genes (BCL2 antagonist/killer 1 and activation of caspase-3). Although the maturation and cleavage rates were similar in all groups, the total cell number per blastocyst and the percentage of apoptotic cells at the blastocyst stage were higher and lower, respectively, in the control and ICA-5 groups than in the aging group. Conclusion: ICA protects oocytes against damage during aging in vitro; therefore, it can be used to improve assisted reproductive technologies.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.68-68
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• 2001

Blastocyst formation, consisting of the inner cell mass (ICM) and trophectoderm (TE), is the first differentiation process during embryonic development in mammals. It has been hypothesized that the proportion of ICM to TE in the blastocyst may be crucial for subsequent developmental competence of early embryos, which it may be expressed as a sensitive indicator for evaluating in vitro systems. In this study ICM/total cell ratio of nuclear transfer (NT) embryos was compared with IVF-derived and in vivo embryos. Somatic cell nuclei obtained from a fetus at Day 40 of gestation were transferred into the enucleated oocyte and then cultured in NCSU 23 medium for 6 days as previously described (Koo et al., Biol. Reprod. 2000; 63:986-992). ICM and TE cells of blastocysts were determined by using a differential staining method (Han et al., Biol. Reprod. 1999; 60:1110-1113). Development rate (9.8$\pm$2.5%, 23/225) to the blastocyst stage of NT embryos was lower than IVF embryos (23.8$\pm$2.7%, 53/223). Thus, a difference was detected in the in vitro developmental rate to blastocyst stage between NT and IVF-derived embryos (P<0.05). In the next experiment, we investigated ICM and TE nuclei to assess the quality of blastocysts that produced by NT, IVF and in vivo, respectively. NT blastocysts (27.6$\pm$8.3) showed a smaller total cell number than IVF-derived (42.6$\pm$17.4) and in vivo embryos (283.9$\pm$103.5) (P<0.05). Ratios of ICM/total cells in NT, IVF and in vivo blastocysts were 15.1$\pm$ 18.6% (n=56), 12.3$\pm$9.2% (n=57) and 30.4$\pm$6.8% (n=40), respectively. Individual blastocysts for the ratio of ICM/total cells were assigned to 3 groups (I; <20%, II; 20 to 40% and III;>40%). As the results, most in vivo blastocysts (97.5%, 39/40) were distributed into group II while most NT (78.6%, 44/56) and IVF-derived blastocysts (82.5%, 47/57) were allocated to group I. Thus, our data show that NT or IVF-derived embryos have aberrant morphology during early development in vitro systems, suggesting that these anomalies may result in developmental failures of the NT embryos to term.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.80-80
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• 2002

This study was carried out that to investigate the effects of amino acids supplemented with culture medium on development of porcine embryos cultured in vitro. Cumulus oocyte complexes (COCs) were cultured in the maturation medium containing hormones (0.5$\mu\textrm{g}$/$m\ell$ LH, 0.5$\mu\textrm{g}$/$m\ell$ FSH and 1$\mu\textrm{g}$/$m\ell$ estradiol-17${\beta}$) for 20-22 h at 39$^{\circ}C$ in an atmosphere of 5% CO$_2$in air. Subsequently, COCs were cultured in hormone-free maturation medium for 20-22 h. After maturation for 40-44h, oocytes were removed cumulus cells by pipetting and cultured with epididymal sperm for 5 h in the mTBM. Embryos obtained were divided in 4 groups (1) cultured in NCSU 23 containing 0.4% BSA to blastocyst stage(Control), (2) essential amino acids (EA), (3) non-essential amino acids (NA), (4) mixture of essential and non essential amino acid (EA+NA). All treated groups(2-4) were used a glucose free NCSU 23 medium supplemented with pyruvate (0.33 mM), lactate (4.5 mM) to morula stage. From morula to blastocyst stage embryos of all treated groups were cultured in NCSU 23 containing 0.4% BSA. The rates of cleaved oocytes at 48 h after IVF were from 82% to 88% in the groups of control, EA, NA and EA+NA, respectively. The in vitro developmental rates into blastocysts in the groups of EA and EA+NA were significantly (P<0.05) higher than those of group of control (35.1, 35.4 vs. 19.4%, respectively), however, no significant (P<0.05) between control and NA. In conclusion, supplemented with essential amino acid or mixture of essential and non essential amino acid in the culture medium at morula stage increased the rate of development to blastocyst on in vitro produced porcine embryos.