• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.207-211
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• 2010

Current developments in IVF and animal cloning have resulted in increasing demand for large quantities of oocytes and ovarian follicles at specific stages of development. These medical and scientific needs may be met by developing an optimal culture system for preantral follicles. In this study, we investigated the growth of porcine preantral follicle cultures in different media and in the presence and absence of serum. Follicles were manually dissected from ovaries obtained from prepubertal gilts at a local slaughterhouse, and cultured for 3 days in M199 or NCSU23 medium supplemented with porcine FSH, transferrin, L-ascorbic acid and insulin. Follicle diameters were measured on day 1 and 3 of culture. In Experiment 1, the effect of supplementing culture medium with fetal calf serum (FCS) on porcine preantral follicle growth was examined. In the group of cultures supplemented with FCS, follicle diameter after 3 days of culture, survival rate and antrum formation rate in the FCS group were significantly higher than those of the control group. In Experiment 2, the effects of culture medium (M199 and NCSU23) on follicle growth were compared. Follicle diameters were increased in the M199 group, compared with those in NCSU23 (p<0.05), but we observed no significant differences in survival and antrum formation rates between cultures grown in the two media. In conclusion, supplementation of the culture medium with serum enhances preantral follicle growth and antrum formation, and M199 is superior to NUSU23 for porcine preantral follicle culture in vitro.

• Clinical and Experimental Reproductive Medicine
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• v.39 no.3
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• pp.95-106
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• 2012

It has been revealed that multiple cohorts of tertiary follicles develop during some animal estrous cycle and the human menstrual cycle. To reach developmental competence, oocytes need the support of somatic cells. During embryogenesis, the primordial germ cells appear, travel to the gonadal rudiments, and form follicles. The female germ cells develop within the somatic cells of the ovary, granulosa cells, and theca cells. How the oocyte and follicle cells support each other has been seriously studied. The latest technologies in genes and proteins and genetic engineering have allowed us to collect a great deal of information about folliculogenesis. For example, a few web pages (http://www.ncbi.nlm. nih.gov; http://mrg.genetics.washington.edu) provide access to databases of genomes, sequences of transcriptomes, and various tools for analyzing and discovering genes important in ovarian development. Formation of the antrum (tertiary follicle) is the final phase of folliculogenesis and the transition from intraovarian to extraovian regulation. This final step coordinates with the hypothalamic-pituitary-ovarian axis. On the other hand, currently, follicle physiology is under intense investigation, as little is known about how to overcome women's ovarian problems or how to develop competent oocytes from in vitro follicle culture or transplantation. In this review, some of the known roles of hormones and some of the genes involved in tertiary follicle growth and the general characteristics of tertiary follicles are summarized. In addition, in vitro culture of tertiary follicles is also discussed as a study model and an assisted reproductive technology model.

• Korean Journal of Animal Reproduction
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• v.21 no.2
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• pp.117-122
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• 1997

The aim of this study was to investigate the effect of the follicular culture from which the oocytes originate on their subsequent in vitro maturation ability. Ovarian follicles were isolated and cultured according to size(1~2mm, 2~6mm and 6~8mm) for 42~44 h. The rates of germinal vesicle breakdown(GVBD) in each groups were 87%(65/75), 82%(80/97) and 89%(47/53), but the oocytes maturation were su, pp.essed at anaphase-I stage. In spite of the adding porcine follicular fluid and/or hormones in maturation medium, maturation ability of oocytes from follicle cultured for 21~22 h were inhibited. When oocytes from follicle cultured for 4 h at various temperature were incubated for 38~40 h, the rates of oocytes maturation from follicle cultured at 2$0^{\circ}C$(51%, 26/51) and 39$^{\circ}C$(54%, 26/48) were significant higher(P<0.05) than group cultured at 4$^{\circ}C$(33%, 19/58). On the other hand, the GVBD were stared 2 h after culture of follicle of oocytes. To summairze, oocytes maturation by follicular culture were inhibited at anaphase-I stage in porcine. When the follicle cultured for 4 h, maturation were completed to metaphase-II stage. However, rates of GVBD in oocytes from follicular culture were higher than oocytes cultured in medium.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.629-634
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• 2012

This study was conducted to improve the yield of mature oocytes from in vitro-culture of ovarian primary follicles by optimizing follicle retrieval from neonatal mice of different ages. Primary follicles of 75 to $99{\mu}m$ in diameter were collected daily from 7- to 14-day-old neonatal mice, and subsequently cultured in ${\alpha}$-MEM medium. Number of primary follicles isolated, growth of the follicle during in vitro-culture and maturation of intrafollicular oocytes were monitored. Overall, mean number of preantral follicles per animal was improved from 10.7 to 88.7 as the age of follicle donors was increased from 7 to 14-day-old. Number of primary follicles was increased gradually up to 11-day-old (35.7 follicle per an animal), then reduced to 29 in 14-day-old (p = 0.0013). More follicles retrieved from 10-day-old or 11-day-old females maintained their morphological normality at the end of primary culture than the follicles retrieved from 9-day-old. Of those cultured, primary follicles retrieved from 11-day-old mice yielded largest larger number of early secondary follicles than the follicles retrieved from in the other ages (39 vs. 13 to 29%). More than 3.3-times increase (0.86 to 2.86; p<0.05) in an average number of mature oocytes per animal was observed in the group of 11-day-old, compared with 9-day-old. However, no difference was found in the percentage of primary follicles developing into the pseudoantral stage (21 to 30%; p = 0.5222) and in the percentage of oocytes mucified (32 to 39%; p = 0.5792). In conclusion, a positive correlation between retrieval time and follicle growth was detected, which influences the efficiency to derive mature oocytes by follicle culture.

• Asian-Australasian Journal of Animal Sciences
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• v.22 no.1
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• pp.35-41
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• 2009

This study was undertaken to evaluate how ${\beta}$-mercaptoethanol (bME), an exogenous antioxidant, interacts with preantral follicles cultured in vitro. Mouse primary or secondary follicles were cultured in glutathione (GSH)-free or GSH-containing medium supplemented with bME of various concentrations, and the growth of preantral follicles, the maturation of intrafollicular oocytes and preimplantation development after parthenogenesis were monitored. In experiment 1, 0, 25, 50 or 100 ${\mu}M$ bME was added to culture medium supplemented with 100 ${\mu}M$ GSH or not. When secondary follicles were cultured in GSH-free medium, no significant change in follicle growth was detected after bME addition. However, exposure to bME in the presence of GSH significantly inhibited both follicle growth and oocyte maturation. Such detrimental effect became prominent in primary follicles and bME strongly inhibited follicle growth in the absence of GSH. In conclusion, there are stage-dependent effects of bME on follicle growth and oocyte maturation, and selective use of antioxidants contributes to establishing an efficient follicle culture system.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.1
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• pp.35-39
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• 2019

This study aimed to recover the ovarian function through in vitro culture of preantral follicles from aged mice. First, we isolated the preantral follicles from ovaries of sixty-seven-week old B6D2F1 mice with decreased fecundity to know how many follicles were present in them, which was 6 preantral follicles including 2 primary, 2 early secondary and late secondary follicles from 8 aged mice. It was confirmed that a few follicles (~2) were present in aged mice through histological analysis compared to adult mice as control. The 9 days of in vitro culture of preantal follicles showed in vitro growth and induced maturation after treatment with hCG (2.5 IU/mL) and EGF (5 ng/mL). Cumulus cells in the cumulus-oocyte complexes (COCs) were removed using hyaluronidase and oocytes at the germinal vesicle (GV) and GV breakdown (GVBD) were obtained from preantral follicle culture of aged mice in vitro. In conclusion, these observations demonstrated that there still were a few preantral follicles in the ovaries of 67 week-old mice, which we were able to culture in vitro and oocytes were obtained from them. This study proposed an in vitro culture system using preantral follicle as a therapeutic strategy for fertility preservation in humans for assisted reproductive medicine.

• 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.

• Clinical and Experimental Reproductive Medicine
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• v.48 no.2
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• pp.111-123
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• 2021

Objective: Using domestic cats as a biomedical research model for fertility preservation, the present study aimed to characterize the influences of ovarian tissue encapsulation in biodegradable hydrogel matrix (fibrinogen/thrombin) on resilience to cryopreservation, and static versus non-static culture systems following ovarian tissue encapsulation and cryopreservation on follicle quality. Methods: In experiment I, ovarian tissues (n=21 animals; 567 ovarian fragments) were assigned to controls or hydrogel encapsulation with 5 or 10 mg/mL fibrinogen (5 or 10 FG). Following cryopreservation (slow freezing or vitrification), follicle viability, morphology, density, and key protein phosphorylation were assessed. In experiment II (based on the findings from experiment I), ovarian tissues (n=10 animals; 270 ovarian fragments) were encapsulated with 10 FG, cryopreserved, and in vitro cultured under static or non-static systems for 7 days followed by similar follicle quality assessments. Results: In experiment I, the combination of 10 FG encapsulation/slow freezing led to greater post-thawed follicle quality than in the control group, as shown by follicle viability (66.9%±2.2% vs. 61.5%±3.1%), normal follicle morphology (62.2% ±2.1% vs. 55.2%±3.5%), and the relative band intensity of vascular endothelial growth factor protein phosphorylation (0.58±0.06 vs. 0.42±0.09). Experiment II demonstrated that hydrogel encapsulation promoted follicle survival and maintenance of follicle development regardless of the culture system when compared to fresh controls. Conclusion: These results provide a better understanding of the role of hydrogel encapsulation and culture systems in ovarian tissue cryopreservation and follicle quality outcomes using an animal model, paving the way for optimized approaches to human fertility preservation.

• Journal of Animal Reproduction and Biotechnology
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• v.34 no.2
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• pp.117-122
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• 2019

The objective of this study was to establish an in vitro culture system for ovarian preantral follicles of B6D2F1. First, we optimized the in vitro preantral-follicle culture by culture duration, follicle stimulating hormone (FSH) type, and activin A concentration. Duration of in vitro culture for 9, 11, and 13 days was sufficient for the normal development of preantral follicles to antral follicles. Formation of cumulus cell-oocyte complex (COC) was induced by treatment with human chorionic gonadotropin (hCG; 2.5 IU/mL) and epidermal growth factor (EGF; 5 ng/mL). In addition, metaphase II (MII) oocytes formed during this in vitro culture of preantral follicles. In vitro preantralfollicle culture for 9 days showed higher rates of growth and maturation, thus yielding a greater number of antral follicles, and there were significant differences (p < 0.05) in the number of MII oocytes (that formed from these preantral follicles via differentiation) between the 9-day culture and 11-day or 13-day culture. The follicles cultured for 9 days contained a tightly packed well-defined COC, whereas in follicles cultured for 11 days, the COC was not well defined (spreading was observed in the culture dish); the follicles cultured for 13 days disintegrated and released the oocyte. Second, we compared the growth of the preantral follicles in vitro in the presence of various FSH types. There were no significant differences in the growth and maturation rates and in differentiation into MII oocytes during in vitro culture between preantral follicles supplemented with FSH from Merck and those supplemented with FSH from Sigma. To increase the efficiency of MII oocyte formation, the preantral follicles were cultured at different activin A concentrations (0 to 200 ng/mL). The control follicles, which were not treated with activin A, showed the highest rate of differentiation into antral follicles and into MII oocytes among all the groups (0 to 200 ng/mL). Therefore, activin A (50 to 200 ng/mL) had a negative effect on oocyte maturation. Thus, in this study, we propose an in vitro system of preantral-follicle culture that can serve as a therapeutic strategy for fertility preservation of human oocytes for assisted reproductive medicine, for conservation of endangered species, and for creation of superior breeds.

• Asian-Australasian Journal of Animal Sciences
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• v.20 no.8
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• pp.1190-1195
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• 2007

This study was conducted to improve efficiency of a follicle culture system without reducing developmental competence of intrafollicular oocytes. Preantral follicles (100 to $125{\mu}m$ in diameter) of F1 hybrid (B6CBAF1) mice were cultured singly for 216 h in modified ${\alpha}$-MEM-glutamax medium, to which 2.5 IU/ml hCG and epidermal growth factor was added 16 h prior to the end of culture. Medium change was either performed three times (54 h interval), twice (72 h interval), once (108 h interval), or not at all (216 h interval). Maturation (progression to the metaphase II stage) of intrafollicular oocytes was detected from 4 days after culture in the three-times change treatment, while all treatments yielded mature oocytes from day 5 of culture. Compared with the three-times change, decreasing the change frequency to once did not reduce the capacity to begin maturation (germinal vesicle breakdown of 82 to 86%), to mature (78 to 79%) and to develop into blastocysts after parthenogenetic activation (29 to 32%). Morphological parameters were similar among these treatments. Except for the no medium change treatment, similar colony-forming activity of inner cell mass cells after culturing of blastocysts in leukemia inhibitory factor-containing medium was detected, while the morphology of the colony-forming cells deteriorated in the change-once treatment compared with the change twice or three-times. In conclusion, the efficiency of the preantral follicle culture system could be improved by reducing frequency of medium change up to a 72 h interval (three times in total 216 h culture) without decreasing developmental competence of oocytes.