• Clinical and Experimental Reproductive Medicine
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• v.28 no.2
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• pp.79-86
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• 2001

Objective: Mouse pre-antral follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and/or phygiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Methods: Pre-antral follicles ($91{\sim}120{\mu}m$) were isolated mechanically by fine 30G needles not using enzymes from ovaries of 3-6 week-old female ICR mice. Isolated pre-antral follicles were cultured in $20{\mu}l$ droplets of TCM (n=17; follicles: $107.8{\pm}1.58{\mu}m$; oocytes: $57.9{\pm}1.2{\mu}m$) or MEM (n=12; follicles: $109.3{\pm}2.53{\mu}m$; oocytes: $55.4{\pm}1.6{\mu}m$) under mineral oil on the 60 mm culture dish. All experimental media was supplemented with 10% FBS without Gns and/or physiological factors. Pre antral follicles were individually cultured for 8 days. Antram formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using precalibrated ocular micrometer at X200 magnifications during in vitro culture. Results were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when p<0.05. Results: Antrum formation had started in two culture media on day 2. On day 8, antrum formation had occurred in 58.3% of pre-antral follicles cultured in DMEM, but only in 23.5% of those cultured in TCM (p=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day 4 and 8. On day 4, follicular diameter was similar (p=0.1338) in TCM ($119.4{\pm}2.58{\mu}m$) and MEM ($125.4{\pm}4.52{\mu}m$). However, on day 8, diameters of pre-antral follicles cultured in MEM ($168.9{\pm}17.29{\mu}m$) were significantly bigger (p=0.0248) than that in TCM ($126.7{\pm}4.28{\mu}m$). On day 4 and 8, diameters of intra-follicular oocytes were similar in TCM ($67.1{\pm}1.3$ and $72.4{\pm}0.9{\mu}m$) and MEM ($65.2{\pm}1.7$ and $73.3{\pm}1.5{\mu}m$), respectively. Conclusion: We can conform that medium without Gns and/or physiological factors can be used for in vitro antrum formation and growth of pre-antral follicles and intra-follicular oocytes in mouse. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.8-8
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• 2001

Mouse follicles require the addition of gonadotropins (Gns) to complete maturation and ovulation of oocyte and antrum formation of follicles in vitro. However, we tried examination of in vitro growth of mouse pre-antral follicles in medium without Gns and physiological factors. And also, pre-antral follicles were isolated from ovaries by mechanical method. Our present studies were conducted to evaluate on the growth of follicles and intra-follicular oocytes and antrum formation in vitro of mouse pre-antral follicles in two different media. Pre-antral follicles (91-120${\mu}{\textrm}{m}$) were isolated mechanically by fine 30G needles not using enzymes from ovary of 3-6 weeks old female ICR mice. Isolated pre-antral follicles were cultured in 20 ${mu}ell$ droplets of TCM (n=17; follicles: 107.8 $\pm$ 1.58 ${\mu}{\textrm}{m}$; oocytes: 59.9$\pm$1.2 ${\mu}{\textrm}{m}$) or MEM (n=12; follicles: 109.3$\pm$2.53 ${\mu}{\textrm}{m}$; oocytes: 55.4 $\pm$1.6${\mu}{\textrm}{m}$) under mineral oil on the 60mm culture dish. All experimental media was supplemented with 10% FBS but without Gns and/or physiological factors. Pre-antral follicles were individually cultured in drops for 8 days. Antrum formation and growth of pre-antral follicles and intra-follicular oocytes were evaluated using a precalibrated ocular micrometer at $\times$200 magnifications during in vitro culture. Results between different groups were analyzed using combination of Student's t-test and Chi-square, and considered statistically significant when P<0.05. Antrum formation of pre-antral follicles had started in two culture media on day-2. On day-8, antrum formation had occurred in 58.3%(7/12) of pre-antral follicles cultured in MEM, but only in 23.5% (4/17) of those cultured in TCM (P=0.0364). Growth of pre-antral follicles and intra-follicular oocytes were observed on day-4 and -8. On day-4, follicular diameters was similar (P=0.1338) in TCM (119.4$\pm$2.58 ${\mu}{\textrm}{m}$) and MEM (125.4$\pm$4.52 ${\mu}{\textrm}{m}$). However, on day-8, diameters of pre-antral follicles cultured in MEM (168.9$\pm$17.29 ${\mu}{\textrm}{m}$) was significantly (P=0.0248) bigger than that in TCM (126.7$\pm$4.28 ${\mu}{\textrm}{m}$). On day-4 and -8, diameters of intra-follicular oocytes were similar TCM (67.1$\pm$1.3 and 72.4$\pm$0.9${\mu}{\textrm}{m}$) and MEM (65.2$\pm$1.7 and 73.3$\pm$1.5 ${\mu}{\textrm}{m}$), respectively. We can conform that medium not supplemented with Gns and/or physiological factors can be used for in vitro antrum formation and growth of mouse pre-antral follicles and intra-follicular oocytes. In conclusion, MEM supplemented with FBS can be used for growth in vitro of mouse pre-antral follicles isolated mechanically.

• Asian-Australasian Journal of Animal Sciences
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• v.19 no.12
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• pp.1711-1715
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• 2006

To predict the number of preantral (primordial, primary and secondary) follicles retrieved from bovine ovaries, we examined the relationship between morphological parameters of ovaries and number of preantral follicles retrieved mechanically. The preantral follicles were retrieved mechanically by slicing ovarian tissue and the influences of size of the ovaries, number of antral follicles, and presence of cystic follicle and corpus luteum on the retrieval were evaluated. Total 77 ovaries were used and significant (p<0.05) relationship was detected between the number of antral follicles and the presence of cystic follicles, and the retrieval number. More preantral follicles were retrieved from the ovaries having more than 20 antral follicles than those having less than 20 antral follicles (17,760${\pm}$5,637 vs. 3,689${\pm}$537) in the ovarian cortex. The retrieval number was significantly reduced in cystic ovaries compared with non-cystic ovaries (5,167${\pm}$825 vs. 20,631${\pm}$6,507). However, neither ovary size (<3.5, 3.5 to 4.0, 4.0 to 4.5 and >4.5 cm) nor the presence of corpus luteum affected the follicle retrieval. In conclusion, the number of preantral follicles retrieved from the ovaries can simply be predicted by the number of antral follicles and the presence of cystic follicles in the ovarian cortex.

• Nuclear Engineering and Technology
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• v.33 no.3
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• pp.255-260
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• 2001

In mammals, most of the follicles can not be ovulated, and instead, are degenerated throughout the entire reproductive period. However, the precise mechanism of follicle atresia is unknown. Three weeks old female mice (ICR strain) were ${\gamma}$-irradiated with a dose of LD$^{50}$ . Before irradiation (day 0) and at day 1, 2, and 3 after irradiation, the normal and atretic preantral and antral follicles of the left ovaries were morphologically observed. Atretic follicles at 2 days after irradiation had numerous cell debris, apoptotic cells and bodies, and polymorphonuclear leukocytes in the antral cavity. In severely atretic follicles, numerous polymorphonuclear leukocytes infiltrated into the follicle. The frequencies of atretic antral (58.0 $\pm$8.6) and preantral follicles (27.3$\pm$11.2) induced by ${\gamma}$-radiation increased to 94.0$\pm$3.4 and 86.9$\pm$7.6, respectively at 2 days after irradiation (p<0.05). The number of follicles with one or more neutrophils in the largest cross sections at 2 and 3 days after irradiation significantly increased (p<0.05). It can be concluded that ${\gamma}$-radiation triggers the recruitment of neutrophils into the follicles during degeneration. The ovarian follicles can make a role of informative biological end-point useful for disaster-prevention.

• Reproductive and Developmental Biology
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• v.37 no.1
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• pp.17-22
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• 2013

Although assisted reproductive technology is very useful to develop novel and therapeutic biomaterials for reproduction, research on molecular mechanism of folliculogenesis in pig is not clear. Therefore, the alteration of gene expression during follicular development in pigs was examined in this study. The expression of folliculogenesis-related genes was quantified in preantral ($250{\sim}300{\mu}m$) and antral (> $300{\mu}m$ in diameter) follicles, and overall gene expression was evaluated by a genome-wide microarray. The microarray results showed that 219 genes were differentially expressed, and of those, 10 and 22 known genes showed higher and less expression at the preantral stage than at antral stages, respectively. Among them, the expression of NR0B1, PPARG, GATA4, and ANXA2 genes related to folliculogenesis was validated by quantitative real-time PCR analysis. The expression of PPARG and GATA4 genes were increased at antral stages, but a significantly stage-specific increase (p<0.05) was only detected in annexin A2 (ANXA2) in antral-stage follicles. The expression of NR0B1 genes was increased at preantral stage and these patterns of gene expression were comparable to the results obtained by microarray analysis. We propose that the systematical regulation of genes supporting specific follicle stage should be employed for improved in-vitro folliculognesis.

• Journal of Animal Science and Technology
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• v.60 no.6
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• pp.11.1-11.7
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• 2018

After pubertal, cohort of small antral follicles enters to gonadotrophin-sensitive development, called recruited follicles. This study was aimed to identify candidate genes in follicular cyclic recruitment via analysis of protein-protein interaction (PPI) network. Differentially expressed genes (DEGs) in ovine granulosa cells of small antral follicles between follicular and luteal phases were accumulated among gene/protein symbols of the Ensembl annotation. Following directed graphs, PTPN6 and FYN have the highest indegree and outdegree, respectively. Since, these hubs being up-regulated in ovine granulosa cells of small antral follicles during the follicular phase, it represents an accumulation of blood immune cells in follicular phase in comparison with luteal phase. By contrast, the up-regulated hubs in the luteal phase including CDK1, INSRR and TOP2A which stimulated DNA replication and proliferation of granulosa cells, they known as candidate genes of the cyclic recruitment.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.4
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• pp.347-351
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• 2021

Objective: We investigated the impact of vitamin D₃ (VD3) supplementation during mouse preantral follicle culture in vitro and the mRNA expression of 25-hydroxylase (CYP2R1), 1-alpha-hydroxylase (CYP27B1), and vitamin D receptor (VDR) in mouse ovarian follicles at different stages. Methods: Preantral follicles were retrieved from 39 BDF1 mice (7-8 weeks old) and then cultured in vitro for 12 days under VD3 supplementation (0, 25, and 50 pg/mL). Follicular development and the final oocyte acquisition were assessed. Preantral follicles were retrieved from 15 other BDF1 mice (7-8 weeks old) and cultured without VD3 supplementation. Three stages of mouse ovarian follicles were obtained (preantral, antral, and ruptured follicles). Total RNA was extracted from the pooled cells (from 20 follicles at each stage), and then reverse transcriptase-polymerase chain reaction was performed to identify mRNA for CYP2R1, CYP27B1, and VDR. Results: The survival of preantral follicles, rates of antrum formation and ruptured follicles (per initiated follicle) and the number of total or mature oocytes were all comparable among the three groups. Both CYP2R1 and CYP27B1 were expressed in antral and ruptured follicles, but not in preantral follicles. VDR was expressed in all three follicular stages. Conclusion: VD3 supplementation in vitro (25 or 50 pg/mL) did not enhance mouse follicular development or final oocyte acquisition. Follicular stage-specific expression of CYP2R1, CYP27B1, and VDR was observed.

• Animal Bioscience
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• v.37 no.6
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• pp.1007-1020
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• 2024

Objective: We increased the nuclear maturation rate of antral follicle derived oocytes by using a pre-in vitro maturation (IVM) culture system and improved the developmental potential of these porcine pathenotes by supplementing with melatonin. Furthermore, we investigated the expression patterns of genes involved in cumulus expansion (HAS2, PTGS2, TNFAIP6, and PTX3) derived from small and medium antral follicles before and after oocyte maturation. Methods: Only the cumulus oocyte-complexes (COCs) derived from small antral follicles were induced with [Pre-SF(+)hCG] or without [Pre-SF(-)hCG] the addition of human chorionic gonadotropin (hCG) during the last 7 h of the pre-IVM period before undergoing the regular culture system. The mature oocytes were investigated on embryonic development after parthenogenetic activation (PA). Melatonin (10^-7 M) was supplemented during in vitro culture (IVC) to improve the developmental potential of these porcine pathenotes. Results: A pre-IVM culture system with hCG added during the last 7 h of the pre-IVM period [Pre-SF(+)hCG] effectively supported small antral follicle-derived oocytes and increased their nuclear maturation rate. The oocytes derived from medium antral follicles exhibited the highest nuclear maturation rate in a regular culture system. Compared with oocytes cultured in a regular culture system, those cultured in the pre-IVM culture system exhibited considerable overexpression of HAS2, PTGS2, and TNFAIP6. Porcine embryos treated with melatonin during IVC exhibited markedly improved quality and developmental competence after PA. Notably, melatonin supplementation during the IVM period can reduce and increase the levels of intracellular reactive oxygen species (ROS) and glutathione (GSH), respectively. Conclusion: Our findings indicate that the Pre-SF(+)hCG culture system increases the nuclear maturation rate of small antral follicle-derived oocytes and the expression of genes involved in cumulus expansion. Melatonin supplementation during IVC may improve the quality and increase the blastocyst formation rate of porcine embryos. In addition, it can reduce and increase the levels of ROS and GSH, respectively, in mature oocytes, thus affecting subsequent embryos.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.243-243
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• 2004

Purpose: This study was conducted to obtain the relationship between initial size of pre-antral follicles (PAF) and intra-follicular oocytes (IFO) and their in vitro growth (IVG) in medium without gonadotropins (Gns) using PAF isolated from mouse ovaries mechanically. (omitted)

• Journal of Embryo Transfer
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• v.25 no.2
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• pp.93-96
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• 2010

The isolation of preantral follicles from the ovaries of bovine was performed under mechanical and enzymatic methods. A significant increase in the total number of follicles retrieved was detected when tissue chopper was used. Micro-dissection could supply good quality, larger sized follicles (400 to $700{\mu}m$) but with the lowest yield ($9.0{\pm}1.0$). The isolated preantral and early antral follicles were cultured for 14 days. Follicles isolated by the mechanical method had a greater growth during a culture period than follicles collected enzymatically. Morphologically normal bovine oocytes from early antral follicles after 14 days culture were 59.6% after culture and after 24 h of maturation culture, 12.9% of in vitro-grown oocytes reached the second metaphase. In conclusion, this study showed that mechanical method can be used effectively to isolate intact preantral follicles from bovine ovaries.