Effect of alpha lipoic acid on in vitro development of bovine secondary preantral follicles

Abstract

The present study aimed to evaluate the in vitro effect of alpha lipoic acid (ALA) addition to the culture medium on the development of the bovine secondary preantral follicles. Bovine secondary preantral follicles were collected and divided into two groups depending on their size (80–100 μm and 100–110 μm). They were cultured in vitro for 15 days (D) using different media including at three different doses of ALA. The genes expression levels of follicle-stimulating hormone beta-subunit (FSHR), insulin-like growth factor (IGF-1), Activin, luteinizing hormone/choriogonadotropin receptor, bone morphogenetic protein receptor type IA, transforming growth factor beta receptor 1, growth differentiation factor 9, BCL2-associated X protein (BAX), and C-Myc were studied using real-time polymerase chain reaction. The protein expression levels of FSHR, IGF-1, and BAX were measured using Western blot analysis. The results of the present work revealed that in vitro addition of ALA-induced significant increase in the growth and development of secondary preantral follicles throughout the culture period as compared to control. The FSHR, IGF-1, luteinizing hormone/choriogonadotropin receptor, bone morphogenetic protein receptor type IA, transforming growth factor beta receptor 1, growth differentiation factor 9, and Activin A genes were upregulated in ALA-treated follicles as compared to the control. On contrary, preapoptotic genes BAX and C-Myc were downregulated in treated follicles compared to control ones. The protein levels of FSHR and IGF-1 were highly expressed in treated follicles compared to control; however, BAX protein was downregulated in the treated follicles groups. The addition of ALA to the culture medium enhances secondary preantral follicles development and growth.

Introduction

The total number of mammalian ovarian follicles is determined early in life, and the depletion of this pool leads in turn to reproductive senescence [1]. Dynamics of ovarian follicle development is an interesting scope to endocrinologists and developmental biologists [1]. Therefore, study of follicle development in vivo and in vitro has gained clinical relevance [1], [2]. Follicles development is usually regulated by reproductive cell signals, especially follicle-stimulating hormone receptor (FSHR), inhibin and other signals [3]. Moreover, several growth factors including FGF-2, IGF-I, bone morphogenetic protein 15 (BMP15), growth differentiation factor 9 (GDF9) were expressed during follicles development [4], [5], [6]. Particularly, oocyte-specific growth factors, bone BMP15, and GDF9 play crucial roles in development and fertility of granulosa cells [6].

In addition, BMP15 and GDF9 are candidates of superfamily called “the transforming growth factor h” [4], [7], [8]. Activities of GDF9 linked with secondary to tertiary transitions during the process of the follicular development [9]. Growth differentiation factor 9 and BMP15 as oocyte-derived factors are elaborated in mediating follicle creation and growth [10], [11], [12]. Therefore, these factors are extremely vital for follicular formation and development [13]. Furthermore, oxidative stress is another factor affecting oocytes maturation and fertilization; excess free radicals production may cause damaging effect on oocyte fertilization and embryo development [4], [14]. As well, free radicals may cause oocyte meiotic arrest in the germinal vesicle; induce embryonic developmental arrest and cell death [4], [14]. Therefore, study on the effect of potential roles of antioxidants on development of bovine secondary preantral follicles is an interesting subject.

Alpha lipoic acid (ALA) plays an important role in regulation of mitochondrial function, ALA in its reduced form acts as potent antioxidants and scavenges free radicals [15]. Moreover, ALA maintains thiol groups on intracellular proteins such as ATPases and other vital proteins [16], [17], [18], [19]. Several studies reported that ALA supplementation reduced apoptosis, however, it upregulates growth and antioxidant enzymes in cumulus-oocyte complexes [16], [17], [18], [19]. In this context, ALA supplementation has been implicated in several diseases including infertility [20], [21]. Despite several studies, address the effect of ALA on follicles maturation. Yet, no enough data are available about the expression of follicles regulating genes and apoptotic-induced genes under the effect of ALA, further studies are required.

It is important to mention that growth factors are expressed at different stage during the follicle development. For example, FGF-2 has been shown to promote activation of primordial follicles [19], whereas IGF-I transcripts were low during the primary follicular stage but increased to a maximum in the late preantral and early antral stages [20]. In other words, no prediction could be suggested on the effect of ALA on primordial follicle, and we think that a serious study should be done on this context.

The secondary preantral follicles are a good cellular model which give means for the study of the biology of follicular development, ovulation, and for studying factors that affect secondary preantral follicles development in vitro [14]. Based on aforementioned data addition of ALA may enhance the development, maturation of the bovine secondary preantral follicles. On contrary, ALA may reduce their apoptosis secondary preantral follicles. In this study, effect of ALA of addition on the expressions of follicles development regulating genes (FSHR, luteinizing hormone/choriogonadotropin receptor [LHCGR], IGF-1, BMPR1a, transforming growth factor beta receptor 1 [TGFβR1], TGFβ1, ActRIIB, GDF9, and Activin A) and apoptotic-induced genes (BCL2-associated X protein [BAX] and C-Myc) were estimated in vitro using bovine secondary preantral follicles.