Modulatory Effects of Single and Complex Vitamins on the In Vitro Growth of Murine Ovarian Follicles
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Vitamin is a well-known co-factor for many metabolic processes and its roles in fertility and follicular growth have been studied. Vitamin supplementation is frequently achieved by daily ingestion in the form of a complex capsule. However, the role of single and complex vitamins in in vitro maturation of murine follicles is not fully elucidated.
In this study, we evaluated the effects of two forms of vitamins. Pure L-ascorbic acid, and multi-vitamin (vitamin C + vitamin B complex) was treated at two different concentrations (50 and 100 µg/ml), to pre-puberty murine follicles during in vitro maturation. To determine the specific stage of growth that is affected by treatment with vitamins, the vitamins were treated from day 0, 4, 9, and 13. Growth of each follicle was assessed by measuring diameters of whole expanded area and of the granulosa cells. Expression of follicular and oocyte growth-related genes and the effect of vitamin on the viability of follicles was assessed using senescence associated β-galactosidase staining.
Treatment with vitamins promoted the in vitro growth of murine follicles and the upregulated the expression of granulosa cell- and oocyte-specific genes such as BMP15, Fsh receptor, and GDF9. The proliferation of the granulosa cells was enhanced by the treatment of vitamin. Fifty µg/ml concentration vitamin showed greater effects compared to higher concentration. The viability of in vitro grown follicles was also significantly improved in vitamin-treated follicles. The effects of single L-ascorbic acid and complex vitamin were not significantly different to those of day 4 and day 9 follicles. Vitamins promoted murine follicle development in vitro with different effects on specific growth stage.
Supplementation of vitamins during in vitro maturation of murine follicles is an efficient strategy for in vitro expansion of follicular cells. These results could be customized to the sophisticated culture of follicles retrieved from aged or cancer-survived female that contain smaller number of follicles with reduced potential to develop into mature follicles.
KeywordsVitamins Murine follicle In vitro maturation L-ascorbic acid
The authors would like to express sincere thanks to the technical assistance of Mi Ae Lee, Da Young Song and Se Hoon Park. This research was supported by the grants of Ildong Pharmaceutical Co. Ltd (0620154170), Ministry of Science and ICT (2016R1E1A1A01943455) and Seoul National University Hospital (0320170280), Republic of Korea.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
The protocols for animal experiments were previewed and approved by the IACUC of Seoul National University Hospital (IACUC No. 18-0029-S1A0) and all experimental procedures were performed under the regulation of Seoul National University Hospital, Department of Experimental Animal Research Unit.
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