Research articleEffect of alpha lipoic acid on in vitro development of bovine secondary preantral follicles
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.
Section snippets
Materials and methods
Medium 199 with Earle's salts (M199), Dulbeccos's phosphate-buffered saline (PBS), and trypsin with EDTA solution were obtained from Gibco (Grand Island, NY, USA). The present reagents and chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
Results
The cultures were started by freshly healthy isolated follicles and were examined and selected using an inverted microscope. The selected follicles were categorized into two sets according to their size (The first set started the cultures by 80–100 μm and the second by 100–110 μm). Generally, the follicular number and size were increased significantly (P < 0.05) during the in vitro culture in ALA-treated groups (250 and 500 μM) as compared to the others cultures without ALA (control group). The
Discussion
It has been reported that the growth and survival of secondary preantral follicles depend on the presence and concentration of growth signals [22], [23]. Moreover, several studies found that addition of ALA to follicular culture media improved development of secondary preantral follicles [24], [25]. Further studies are required to confirm this factor on secondary follicles maturation. Therefore, the present study was intended to test the effect of in vitro addition of various concentrations of
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University, Saudi Arabia for funding the work through the research group project No. RGP-238.
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