Abstract
BackgroundrhFSH and rhActA have been used in mammalian ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through MAPK and Akt pathways. The aim of our study was to determine the effects of rhFSH and rhActA on Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels in bovine ovarian cortical strips cultured in vitro.MethodsOvarian cortical strips from heifers were cultured in the presence of rhFSH (50 ng/mL), rhActA (100 ng/mL) or combination of these factors for 6 days. The strips were embedded in paraffin for histological observations and homogenized for western blot to determine Akt, pAkt, MAPK1/3 and pMAPK1/3 protein levels after the culture. Determination of primordial, primary and secondary follicle proportions at the end of culture as well as comparison of healthy follicle for each developmental stage after the culture was performed to quantify follicle survival and activation.ResultspAkt protein levels were significantly lower in rhFSH + rhActA group among the other groups, whereas pMAPK1/3 levels were not significantly changed. Follicular activation and survival was measured to be significantly lower in rhFSH + rhActA group. Percentage of healthy primordial follicles was higher in control group whereas healthy secondary follicle proportion was higher in both rhActA and rhFSH groups. rhActA alone had a better impact on follicular activation, since the percentage of the secondary follicles was significantly higher than other treatment groups.ConclusionsThe use of rhActA and rhFSH alone or in the combined form results in differential levels of Akt and MAPK proteins. Both rhActA and rhFSH alone has a remarkable contribution in survival and activation of the follicles in accordance with higher levels of these proteins. Thus, the manipulation of Akt and MAPK pathways with appropriate activators might contribute to proper activation and development of ovarian follicles in vitro.
Highlights
RhFSH and rhActA have been used in mammalian ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through Mitogen activated protein kinases (MAPK) and Akt pathways
Primary and secondary follicles were evaluated at the end of 6 days of culture via histological observations (Fig. 2), it was found that rhActA group had significantly highest and rhFSH + rhActA group had Phosphorylated Akt protein levels in cultured bovine cortical strips Phosphorylated Akt protein level of fresh bovine cortical strips was higher when compared to the culture groups
Akt is known to be phosphorylated at two sites: the Thr308 residue phosphorylated by the phosphoinositide-dependent kinase 1 (PDK1) [45]; the Ser473 residue phosphorylated by the mammalian target of rapamycin [46] as well as integrin-linked kinase (ILK) [47] and mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK2) [48]
Summary
RhFSH and rhActA have been used in mammalian ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through MAPK and Akt pathways. Follicular survival and activation is promoted by stimulation of intracellular phosphoinositide 3-kinase (PI3K)/Akt signaling pathway by Kit ligand in primordial oocytes [6, 7]. The PI3K/Akt signaling pathway is reported to have roles in primordial follicle activation as well as stem cell maintenance and organogenesis [8]. Akt is localized in granulosa cells of primordial follicles and in the basal layers of the granulosa cells of preantral and antral follicles
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