Tepekoy Filiz, Akkoyunlu Gokhan
Department of Histology and Embryology, Faculty of Medicine, Akdeniz University, 07070 Campus, Antalya, Turkey.
J Ovarian Res. 2016 Mar 11;9:13. doi: 10.1186/s13048-016-0222-2.
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. 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.
Ovarian 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.
pAkt 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.
The 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.
重组人促卵泡激素（rhFSH）和重组人激活素A（rhActA）已用于哺乳动物卵巢卵泡培养系统，以在体外激活卵泡生长，并被认为通过丝裂原活化蛋白激酶（MAPK）和蛋白激酶B（Akt）信号通路维持原始卵泡的存活。本研究旨在确定rhFSH和rhActA对体外培养的牛卵巢皮质条中Akt、磷酸化Akt（pAkt）、MAPK1/3和磷酸化MAPK1/3（pMAPK1/3）蛋白水平的影响。
将来自小母牛的卵巢皮质条在rhFSH（50 ng/mL）、rhActA（100 ng/mL）或这些因子的组合存在下培养6天。将皮质条包埋在石蜡中进行组织学观察，并匀浆用于蛋白质免疫印迹，以测定培养后Akt、pAkt、MAPK1/3和pMAPK1/3蛋白水平。在培养结束时测定原始卵泡、初级卵泡和次级卵泡的比例，并比较培养后每个发育阶段的健康卵泡，以量化卵泡的存活和激活情况。
在其他组中，rhFSH + rhActA组的pAkt蛋白水平显著降低，而pMAPK1/3水平无显著变化。rhFSH + rhActA组的卵泡激活和存活率显著降低。对照组中健康原始卵泡的百分比更高，而rhActA组和rhFSH组中健康次级卵泡的比例更高。单独使用rhActA对卵泡激活有更好的影响，因为次级卵泡的百分比显著高于其他治疗组。
单独或联合使用rhActA和rhFSH会导致Akt和MAPK蛋白水平的差异。单独使用rhActA和rhFSH均对卵泡的存活和激活有显著贡献，这与这些蛋白的较高水平一致。因此，用适当的激活剂操纵Akt和MAPK信号通路可能有助于体外卵巢卵泡的正常激活和发育。
