Background
Poor oocyte quality limits the efficiency of in vitro embryo production in cattle and women [
1]. Our previous studies demonstrated a positive association between the transcript abundance of follistatin and oocyte developmental competence using the prepubertal calf and brilliant cresyl blue screening models [
2,
3]. We also found that maternally derived follistatin is essential for bovine early embryonic development and exogenous supplementation of follistatin during first 72 h (h) of in vitro embryo culture has a stimulatory effect on early cleavage, embryonic development to 8- to 16-cell and blastocyst stages and blastocyst cell lineage allocation [
4]. The functional requirement of follistatin in oocyte and early embryonic development has been described in bovine and other mono-ovulatory species [
5].
Follistatin is a transforming growth factor β (TGF-β) superfamily binding protein that may exert its embryotrophic effects through modulation of one or more of the SMAD [
6,
7] or non-SMAD [
8‐
10] signaling pathways. We previously demonstrated that the embryotrophic actions of follistatin on development to 8- to 16-cell and blastocyst stages are linked to the SMAD signaling pathways [
11,
12]. However, how follistatin promotes early embryonic development, particularly early cleavage, is still not fully understood.
Follistatin was first identified as a high affinity Activin binding protein [
13] and its binding affinity to select bone morphogenetic proteins (BMPs) has also been established [
14,
15]. Both BMPs [
16] and Activin [
17] are major regulators of AKT signaling pathway that promotes cell cycle progression during G2/M transition. Studies have shown that AKT stimulates the transition from metaphase I (MI) to metaphase II (MII) in bovine [
18], porcine [
19], mouse [
20,
21] and Xenopus [
22] oocytes by activation of Phosphodiesterase 3A (PDE3A) and cyclin dependent kinase 1 (CDK1) required for resumption and regulation of meiosis. AKT was primarily identified as a serine/threonine specific protein kinase that functions downstream of Phosphatidylinositol 3-kinase (PI3K) [
23]. Inhibition of AKT with a synthetic small molecule inhibitor (AKT inhibitor III) resulted in arrest of bovine oocytes at MI stage [
18]. AKT plays an important role during entry of one cell mouse embryos into the first mitosis as inhibition of AKT resulted in arrest of cell cycle in G1 and G2 phases [
24]. Moreover, inhibition of AKT activity compromised the development of mouse embryos to the blastocyst stage [
25,
26]. Considering the role of AKT in cell cycle progression in oocytes and embryos, we hypothesized that AKT signaling is required for bovine early embryonic development and is linked to the embryotrophic actions of follistatin during in vitro embryo culture. To test this hypothesis, we determined the functional role of AKT in bovine early embryonic development and investigated the relationship between AKT signaling and the embryotrophic actions of follistatin. Effects of treatment with the AKT inhibitors III and IV on development of early bovine embryos and AKT phosphorylation were analyzed. Effects of exogenous follistatin supplementation on AKT phosphorylation and developmental potential of AKT inhibitor treated embryos were also investigated.
Discussion
A growing body of evidence supports an important intrinsic role for follistatin in bovine oocyte quality and early embryo developmental progression in vitro [
3,
4,
11,
12]. While follistatin is best known for its ability to bind and inhibit activity of select TGF-β superfamily ligands, the intrinsic ligands and signaling pathways linked to trophic actions of follistatin on early embryos are not known. Results of the present study demonstrated that pharmacological inhibition of AKT signaling pathway in early bovine embryos reduced their developmental capacity as assessed by early cleavage, total cleavage and rates of development to 8–16-cell and blastocyst stages. Follistatin supplementation during the first 72 h of in vitro embryo culture rescued the adverse effects of AKT signaling inhibition on embryo development and increased the level of AKT phosphorylation, suggesting a potential role for follistatin in regulation of AKT signaling in early bovine embryos. Results demonstrate follistatin regulation of non-canonical TGF-beta superfamily signaling in bovine embryos and a functional link to developmental progression.
AKT is serine/threonine-specific protein kinase that regulates crucial cellular processes such as glucose metabolism, transcription, cellular growth and proliferation [
30‐
32]. Full activation of AKT requires translocation to the plasma membrane and phosphorylation at Thr308 and Ser473 motifs [
33]. Previous studies have established a functional role of AKT during oocyte maturation in various species including bovine [
18] and in early embryogenesis in mouse and pigs [
19,
20,
34]. In bovine embryo, activation of PI3K/AKT pathway by insulin like growth factor − 1 (IGF-1) has been shown to block heat shock induced apoptosis [
35]. In addition, pharmacological inhibition of PI3K, upstream kinase of AKT, using LY294002 resulted in significant decrease in total cleavage, 8 cells and blastocyst development rates in bovine [
36]. In the present study, we used two different pharmacological inhibitors of AKT activity to investigate its role in early bovine embryo development. Pharmacological inhibition was selected over siRNA mediated knockdown because inhibitors are membrane permeable and can function immediately by specifically targeting AKT activity without affecting its total protein abundance in embryo. As inhibitors can function in reversible manner, this allowed us to inhibit AKT signaling for define period (d1–3) of in vitro embryo culture in this study. Our does dependent study showed that AKT inhibitor III at 75 μm or AKT inhibitor IV at 3.5 μM doses exhibited about 50% reduction in AKT phosphorylation levels and therefore were selected as most effective dose to inhibit AKT signaling. These results were in accordance with the previously published studies in bovine and mice [
18,
19,
21]. In addition, higher doses of AKT inhibitor III used in this study has been previously reported to have no nonspecific effect on other kinases downstream to PDK1 [
18].
Our results showed that inhibition of AKT during first 3 days of in vitro embryo culture (before zygotic genome activation) resulted in pronounced reduction in multiple indices of early embryonic development. Early embryonic cleavage at 30 hpi was not detected for all tested doses of AKT inhibitor III. Total cleavage, rates of development to 8- to 16-cell and day 7 blastocyst rates were significantly reduced in embryos treated with the highest dose of AKT inhibitor III. We observed similar effects of AKT signaling inhibition on embryo development using AKT inhibitor IV. Data from other species [
37,
38] suggest that AKT is implicated in regulation of mitotic cell cycle transition from G2 to M phase through activation of M-phase promoting factor (MPF). Therefore, upon inhibition of AKT, the MPF complex remains inactive and the cells are arrested in G1 or G2 stages of the cell cycle [
24,
39]. Reduction of AKT phosphorylation/activity may delay cell cycle progression [
40] or compromise the developmental capacity of treated embryos. Therefore, early cleavage at 30 hpi was not detected, and total cleavage, development to 8- to 16-cell and blastocyst stages were reduced compared with untreated embryos. Collectively, these results demonstrated that inhibition of AKT signaling using two different AKT inhibitors similarly reduced the development of early bovine embryos in vitro suggesting an important functional role of AKT signaling in early bovine embryogenesis. However, further mechanistic studies are required to conclusively elucidate how the AKT signaling pathway regulates early embryonic development in bovine.
We previously established a positive association between follistatin and oocyte developmental competence [
2,
3]. Supplementation of exogenous follistatin during in vitro embryo culture has embryotropic effects in bovine and non-human primates [
4,
41]. Although, follistatin was originally identified as an activin antagonist [
13], the embryotropic actions of follistatin are presumably not mediated by inhibition of activin action as exogenous supplementation of activin mimicked the embryotrophic effects of follistatin [
4]. As a TGF-β superfamily growth factor binding protein, follistatin may exert its embryotrophic actions by modulating SMAD dependent (SMAD1/5/8 or SMAD2/3) or non
-SMAD dependent (AKT, ERK, P38 and JNK) pathways implicated in signaling by this growth factor family [
8]. Previous studies in our laboratory demonstrated that, exogenous follistatin supplementation rescued the effects of SMAD2/3 and SMAD4 inhibition/knockdown on early cleavage but not the effects on development to 8- to 16-cell and blastocyst stages, suggesting that the embryotrophic effects of follistatin on early cleavage are SMAD independent [
11,
12]. In the current study, the effects of follistatin supplementation on development of AKT inhibitor treated embryos were investigated. Treatment with 10 ng/ml follistatin during the initial 72 h of in vitro embryo culture reversed the inhibitory effects of AKT inhibitor III on early cleavage, total cleavage and development to the 8- to 16-cell stage and partially rescued development to the blastocyst stage. Results also demonstrated that AKT inhibitors significantly reduced the levels of AKT phosphorylation in the absence of follistatin. In addition, follistatin treatment not only reverse the AKT inhibitors effect on phosphorylation level but also increase the AKT phosphorylation when supplemented independently for 24 h. These results suggest that follistatin may be playing a role in returning the AKT phosphorylation to the levels required for rescuing the adverse effect of AKT inhibitors on early cleavage. Collectively, results suggest a potential functional link between follistatin action and AKT signaling linked to its embryotropic actions in bovine embryos. As the effects of other signaling pathways can’t be disregarded, further studies may be required to elucidate the effects of inhibition of multiple signaling pathways on the development of early bovine embryos and to dissect the specific effects of each signaling pathway.
Effects of TGF-β superfamily stimulation on AKT signaling appear to be pleiotropic and cell type dependent. It has been demonstrated that members of TGF-β superfamily activate PI3K pathway through phosphorylation of its downstream effector AKT [
42‐
44]. TGF-β can induce a physical interaction between the p85 regulatory subunit of PI3K and the TβRII and TβRI receptors converting phosphatidylinositol-4,5-bisphosphate (PIP
2) to phosphatidylinositol-3,4,5-triphosphate (PIP
3) and subsequently AKT phosphorylation [
45‐
47]. Studies on somatic cell lines indicate that follistatin treatment activates PI3K/AKT signaling pathway [
48,
49]. In the present studies, follistatin treatment reversed the inhibitory effects of AKT inhibitor treatment on AKT phosphorylation levels at 10 h post treatment, but a follistatin dependent increase in basal AKT phosphorylation was not observed in the absence of AKT inhibitor treatment. However, follistatin treatment did increase basal AKT phosphorylation levels 24 h post treatment administration. The exact reason for time dependent effects on AKT signaling observed in response to follistatin treatment are not known but may be reflecting time/treatment induced differences in endogenous growth factor milieu and bioaffinity for specific ligands. Results suggest that follistatin plays a role in regulation of AKT signaling in early bovine embryos. However, AKT seems to not be required for the embryotrophic actions of follistatin.
Conclusions
Results of the present study demonstrate that inhibition of AKT signaling reduce the developmental competence of early bovine embryos suggesting a potential requirement of AKT signaling for bovine early embryonic development. Follistatin supplementation rescued the developmental competence of AKT inhibitor treated embryos suggesting that the examined embryotrophic effects of follistatin do not require AKT signaling. However, follistatin supplementation increased the relative abundance of pAKT in AKT inhibitor treated embryos and increased the level of AKT phosphorylation when supplemented without AKT inhibitor, suggesting that follistatin plays a role in regulation of AKT signaling in early bovine embryos. Together, the results reported here, provide additional insights into understanding the regulation of early embryonic development and the mechanism of action of the embryotrophic agent, follistatin, in early bovine embryos.