Cumulus cells steroidogenesis is influenced by the degree of oocyte maturation

BACKGROUND: The possibility to predict the ability of a germ cell to properly sustain embryo development in vitro or in vivo as early as possible is undoubtedly the main problem of reproductive technologies. To date, only the achievement of nuclear maturation and cumulus expansion is feasible, as all the studies on cytoplasmic maturation are too invasive and have been complicated by the death of the cells analyzed. The authors studied the possibility to test the cytoplasmic quality of pig oocytes by evaluating their ability to produce steroidogenesis enabling factor(s). To this aim, oocytes matured under different culture conditions that allowed to obtain gradable level of cytoplasmic maturation, were used to produce conditioned media (OCM). The secretion of the factor(s) in conditioned media was then recorded by evaluating the ability of the spent media to direct granulosa cells (GC) steroidogenesis. METHODS: In order to obtain germ cells characterized by a different degree of developmental competence, selected pig oocytes from prepubertal gilts ovaries were cultured under different IVM protocols; part of the matured oocytes were used to produce OCM, while those remaining were submitted to in vitro fertilization trials to confirm their ability to sustain male pronuclear decondensation. The OCM collected were finally used on cumulus cells grown as monolayers for 5 days. The demonstration that oocytes secreted factor(s) can influence GC steroidogenesis in the pig was confirmed in our lab by studying E(2 )and P(4 )production by cumulus cells monolayers using a radioimmunoassay technique. RESULTS: Monolayers obtained by growing GC surrounding the oocytes for five days represent a tool, which is practical, stable and available in most laboratories; by using this bioassay, we detected the antiluteal effect of immature oocytes, and for the first time, demonstrated that properly matured germ cells are able to direct cumulus cells steroidogenesis by inhibiting E(2 )production (P < 0.01). Nevertheless, only fully competent oocytes were able to suppress estrogens production, while those cultured under unfavourable conditions were unable to exert any inhibitory effect on the functions of cumulus cells (P < 0.01). CONCLUSION: These results demonstrated that good quality oocytes can be easily selected on the basis of their ability to affect granulosa cell steroidogenesis thus reducing failures in reproductive technologies due to the transfer of fertilized oocytes with a scarce ability to sustain embryo development

Effect of Estradiol and Progesterone on ovine Amniotic Epithelial Cells

This study was designed to clarify Estradiol (E2) and Progesterone (P4) steroid effects on ovine Amniotic Epithelial Cells (oAECs) that has a conserved plasticity and highly self-renewable capacity (Parolini et al., Stem Cells, 26(2), 300–311, 2008; Barboni et al., Stem Cell Rev Rep, 10:725–741, 2014). Based on their conserved immunomodulatory properties, oAECs are suitable for allo and xeno-transplantation (Barboni et al., Cell Transplant, 21(11), 2377–2395, 2012; Muttini et al., Res Vet Sci, 94(1),158–169, 2013). To date, no information is present on the effects of prolonged steroid exposition on AECs. oAECs were cultured as previously reported (Barboni et al., Cell Transplant. 21(11), 2377–2395, 2012) and treated with 12.5μM and 25μM of E2 or P4 (Sigma-Aldrich, Milan, Italy), alone and in both combinations, for three passages. Untreated cells were marked control (CTR). At 70% confluency, cells were detached for doubling time (DT) evaluation. Cells at fourth passage were differentiated for 21 days in osteogenic media (DM) (Mattioli et al., Cell Biol Int 36(1):7-19, 2012) without steroid. Alizarin Red and Alcian-Blue (Sigma-Aldrich, Milano, Italy) stainings were performed. RNA and cDNA were obtained as previously reported (Barboni et al., Cell Transplant. 21(11), 2377–2395, 2012). Real Time for NANOG, SOX2 ,OCT4 stemness genes expression were performed by SensiFast SYBR (Bioline, Aurogene, Rome, Italy) using specific primers (Mattioli et al., Cell Biol Int. 36(1):7-19, 2012).The protocol was: 5 min at 95°C, 30 cycles at 95°C for 15 sec, 60°C for 30 sec, 72°C for 15 sec. Comparative Ct 2-ΔΔC(t) normalization to GAPDH was applied. IHC analyses were carried out for Cytokeratin 8 and αSMA expression as previously reported (Barboni et al. PLoS ONE 7(2): e30974, 2012). Data expressed as mean (±SD), compared by one-way ANOVA followed by Tukey’s test (GraphPad Prism 5). Significant values for P &lt; 0.05. Steroids treated ovine AECs proliferate with significant differences between concentrations. While P4 treated cells showed cuboidal shape and Cytokeratin expression until third passage, CTR and E2 treated cells showed a rapid downregulation of Cytokeratin and increased αSMA expression. oAECs with E2+P4 showed both cell type morphology. Steroids modified stemness genes based on the concentration. 12.5 μM E2, 25μM P4 and 25μM of both E2+P4 treatments maintained higher OCT4, NANOG and SOX2 expressions in treated cells despite their progressive downregulation in the CTR. Moreover, compared to CTR, after Alizarin staining, steroid pretreated cells suffered morphological changes under DM acquiring Alcian Blue-positive chondrogenic-like morphology. AECs stemness properties and plasticity can be modified by prolonged steroidal treatment. These data improve our knowledge, opening new prospective on oAEC use in stem cell-based therapy. Acknowledgments. Research supported by H2020-MSCA ITN EJD-REP BIOTECH 675526

Effects of P4 Antagonist RU486 on VEGF and Its Receptors' Signaling during the In Vivo Transition from the Preovulatory to Periovulatory Phase of Ovarian Follicles

The development of an adequate blood vessel network is crucial for the accomplishment of ovarian follicle growth and ovulation, which is necessary to support the proliferative and endocrine functions of the follicular cells. Although the Vascular Endothelial Growth Factor (VEGF) through gonadotropins guides ovarian angiogenesis, the role exerted by the switch on of Progesterone (P4) during the periovulatory phase remains to be clarified. The present research aimed to investigate in vivo VEGF-mediated mechanisms by inducing the development of periovulatory follicles using a pharmacologically validated synchronization treatment carried out in presence or absence of P4 receptor antagonist RU486. Spatio-temporal expression profiles of VEGF, FLT1, and FLK1 receptors and the two major MAPK/ERKs and PI3K/AKT downstream pathways were analyzed on granulosa and on theca compartment. For the first time, the results demonstrated that in vivo administration of P4 antagonist RU486 inhibits follicular VEGF receptors' signaling mainly acting on the theca layer by downregulating the activation of ERKs and AKTs. Under the effect of RU486, periovulatory follicles' microarchitecture did not move towards the periovulatory stage. The present evidence provides new insights on P4 in vivo biological effects in driving vascular and tissue remodeling during the periovulatory phase