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.

Combined therapy of mesenchymal stem cells with a GLP-1 receptor agonist, liraglutide, on an inflammatory-mediated diabetic non-human primate model.

AIMS: Immunomodulation concurrent with the promotion of ß-cell function is a strategy used to develop innovative therapies for type 1 diabetes (T1D). Here, we assessed the therapeutic potential of co-administration of human clonal mesenchymal stem (stromal) cells (hBM-cMSCs) and liraglutide as a glucagon-like peptide-1 agonist in a non-human primate model with streptozotocin (STZ)-induced diabetes. MAIN METHODS: Diabetes was induced through intravenous (i.v.) multiple low-dose (MLD) infusions of STZ at a dose of 30 mg/kg body weight (b.w.) for five consecutive days, followed by two booster injections of 35 mg/kg on days 12 and 19. After 90 days, the diabetic animals were randomly allocated to two groups: The combination therapy group (n = 4) received injections of 1.5 × 106 hBM-cMSCs/kg b.w. through celiac artery by angiography on days 91 and 105 and daily subcutaneous injections of liraglutide (up to 1.8 mg/day) until day 160 while vehicle group received phosphate-buffered saline. The monkeys were assessed for functional, immunological, and histological analysis. KEY FINDINGS: The combined treatment group had continued reduction in FBG levels up to day 160, which was accompanied by increased b.w., C-peptide, and ß-cell function, and decreased HbA1c and fructosamine levels compared to vehicle group. The combined treatment increased Tregs, IL-4, IL-10, and TGF-ß1 and decreased IL-6 and IL-1ß. Stereological analysis of the pancreatic tissue exhibited more total volume of insulin-secreting islets in the combined treatment group compared to vehicle group. SIGNIFICANCE: Our findings demonstrated this combined treatment impaired the clinical symptoms of diabetes in this animal model through immunomodulation and ß-cell preservation.

Incorporation of arginine, glutamine or leucine in culture medium accelerates in vitro activation of primordial follicles in 1-day-old mouse ovary.

In vitro activation of primordial follicles provides cancer patients subjected to oncotherapy with a safe therapeutic strategy for fertility preservation, however a successful protocol for activation of primordial follicles in prepubertal patients has not yet been defined comprehensively. There is evidence that amino acids such as leucine, arginine and glutamine could stimulate the mammalian target of rapamycin (mTOR) pathway, which plays a pivotal role in primordial follicle activation. Nevertheless, there has been no report that elucidates the effect of these amino acids on in vitro development of ovarian follicles. Therefore, the present study was conducted to evaluate the effects of these amino acids and their combination on the formation and activation of primordial follicles in 1-day-old murine ovaries during an 11-day culture period. The experimental groups consisted of base medium (BM), base medium + arginine (ARG), base medium + glutamine (GLU), base medium + leucine (LEU) and base medium + a combination of arginine, glutamine and leucine (AGL). The proportions of different stages of ovarian follicles and gene expression of regulatory factors were assessed using histology and quantitative real-time PCR on days 5 and 11 of culture. The proportion of transitional and primary follicles was greater in all amino acid-treated groups compared with the BM group (P < 0.05). Moreover, leucine resulted in elevated expression of Gdf9 and Bmp15, and glutamine augmented the expression of Pi3k on day 11 of culture. In conclusion, the present study showed that inclusion of leucine, glutamine, arginine or their combination in the culture medium for murine ovarian tissue could accelerate the activation of primordial follicles and alter the expression of the corresponding factors.