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This study was conducted to investigate the main and interactive effects of two methods of culture medium preparation [base medium vs granulosa cells conditioned medium (GCCM)] and two nutrient supplements [fetal bovine serum (FBS) vs knock-out serum replacement (KSR)] on formation and activation of primordial follicles and gene expression of corresponding factors during a seven-day culture period. One-day-old mouse ovaries were cultured with four different culture media including base medium containing FBS (BMF), base medium containing KSR (BMK), GCCM prepared with FBS (CMF) and GCCM prepared with KSR (CMK), and samples for histological and molecular assessments were collected on days 3 and 7 of culture. Further, steroid content of media was measured. Histological examination showed that KSR enhanced follicular formation and the number of follicular count was greater in BMK than CMF group (P < 0.05). Moreover, follicular activation was higher in CMK group than BMK and CMF groups (P < 0.05). Additionally, RT-PCR revealed that KSR upregulated Gdf9 gene expression (P < 0.05), while GCCM diminished expression of Gdf9, Bmp15, Notch2, Figla and Foxl2 (P < 0.05). GCCM decreased expression of Pten and increased expression of Pi3k (P < 0.05). Besides, hormonal assays indicated higher concentrations of estradiol and progesterone in GCCM compared with base media (P < 0.0001). In conclusion, the present study showed base medium containing KSR could serve as a proper medium for in vitro culture of neonatal mouse ovary since it could better support formation of primordial follicles. Yet BMK did not promote follicular activation as well as GCCM prepared with KSR did, and therefore, requires modifications., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2022 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier B.V. All rights reserved.)

Fertility preservation of prepubertal girls subjected to invasive cancer therapy necessitates defining protocols for activation of isolated primordial follicles. Granulosa (GCs) and cumulus cells (CCs) play pivotal role in oocyte development. Although GCs and CCs share some similarities, they differ in growth factors production. The current study was conducted to evaluate the effects of GCs, CCs and their conditioned media on mice primordial follicles activation. One-day-old mice ovaries were subjected to 6-day culture with base medium (BM), GC conditioned medium (GCCM), GC coculture (GCCC), CC conditioned medium (CCCM) or CC coculture (CCCC). Follicular growth and primordial to primary follicle transition was observed during 6-day culture, and follicular activation rate tended to be greater in GCCM than other groups (0.05