The effect of FSH and activin A on Akt and MAPK1/3 phosphorylation in cultured bovine ovarian cortical strips

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Activin; Akt; FSH; MAPK.

Fig. 1

Stereomicroscopic images of cortical strips. Control (a), rhFSH (b), rhActA (c), rhFSH + rhActA (d) groups in the beginning of the culture (The cover of the plate was closed during photographing to preserve aseptic conditions). Control (e), rhFSH (f), rhActA (g), rhFSH + rhActA (h) groups at the end of the culture. Arrow: Secondary follicles

Fig. 2

Haematoxylin and eosin staining of fresh and cultured cortical strips. Section from uncultured fresh (a, f), control group of day 6 of culture (b, g), day 6 of culture supplemented with rhFSH (c, h), rhActA (d, i) and rhFSH + rhActA (e, j). Red arrows: Primordial follicles. Blue arrows: Primary follicles. Black arrows: Secondary follicles

Fig. 3

Western blot bands and graphics of mathematical values of ImageJ evaluations of pAkt/Akt protein levels in postnatal mouse ovaries. Different letters mark statistical significance (P < 0.05) (One way anova, Holm Sidak method)

Fig. 4

Western blot bands and graphics of mathematical values of ImageJ evaluations of pMAPK1/3 / MAPK1/3 protein levels in postnatal mouse ovaries. Different letters mark statistical significance (P < 0.05) (One way anova, Holm Sidak method)