Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

CONTEXT: Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. AIMS: The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. METHODS: One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. KEY RESULTS: In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). CONCLUSIONS: Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. IMPLICATIONS: This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

High steroid content in conditioned medium of granulosa cells may disrupt primordial follicles formation in in vitro cultured one-day-old murine ovaries.

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.