Granulosa cells undergo BPA-induced apoptosis in a miR-21-independent manner

The growing incidence of infertility has been a topic of concern for reproductive specialists over the years [1]. Of particular note are the factors that have contributed to poor ovarian function and disrupted embryo development. One of these factors is the rise in environmental chemical pollutants that can interfere with ovarian hormonal performance. Endocrine disrupting compounds (EDCs) represent a large class of these chemical pollutants and are scrutinized for their effects on the female reproductive system [2]. Bisphenols, particularly Bisphenol A (BPA), is a common EDC and plasticizer that makes its way into the body through ingestion, inhalation, and dermal absorption [3] and has been detected in most bodily fluids and tissues, including the ovary where it can be absorbed into the follicular fluid and can be taken up by the local cells, such as oocytes, granulosa cells, and theca cells. It is also an estrogen-like chemical that mimics estrogen activity in the ovary and influences gonadotropin release [4].

BPA has been associated with various chronic diseases including obesity, Alzheimer's disease, cancers, infertility, and reproductive dysfunctions [5], acting through a multitude of different mechanisms, which renders BPA research complex but necessary. In addition to the estrogenic properties, BPA is also assumed to possess oxidative and apoptotic activity by reports of reactive oxygen species accumulations, antioxidant imbalance, increased apoptosis, and extensive changes in apoptosis-related gene expression [1,6]. It disrupts cell-to-cell communication within the ovary, which is crucial for the adequate development of the ovarian follicle [7]. BPA dysregulates oocyte maturation, steroidogenesis, fertilization, and early embryonic cleavage, and interferes with blastocyst formation and subsequent implantation [7,8]. Saleh et al. [9], found increased DNA fragmentation in blastocysts that were derived from BPA-treated oocytes, providing evidence of its potential to induce disruptions in future offspring [10].

BPA induces these effects predominantly via changes in gene expression and therefore affects hundreds of thousands of networks and pathways that result in known diseased phenotypes [11]. More recently, evidence suggests that BPA induces changes in epigenetic mechanisms including noncoding RNAs (ncRNAs), such as microRNAs (miRNA) [[12], [13], [14]]. miRNAs are short ncRNA, that function post-transcriptionally to silence gene expression by preventing translation or inducing mRNA degradation [15]. Transcriptome profiling suggests that miRNAs are extremely susceptible to BPA and target enrichment studies have correlated these changes to the effects observed on classical gene expression [16,17]. Pathway predictions have related this information to disruptions in the ovary, granulosa cells, oocytes, and even the early embryo [17]. An example is a BPA-induced increase in miR-146a in fetal ewe ovaries, which was shown to target steroidogenic genes, associated with disruptions in gonadal differentiation and folliculogenesis [16]. Wistar rats exposed to BPA exhibited increased miR-224 expression, which was correlated with changes in aromatase protein levels, estradiol serum levels, and FSH production resulting in poor phenotypic outcomes including reduced pup weight at birth and decreased anogenital distance [12].

One of the most well-documented damaging effects of BPA on the growing follicle is atresia, whereby the follicle fails to develop, degenerates, and induces follicle death [14]. A rising incidence of follicular atresia is associated with increased maternal age, lower ovulation rates, lower ovarian reserves, and overall reduced fertility [14,18]. Atresia is a complex process comprising several regulatory mechanisms with granulosa cell apoptosis widely accepted as the hallmark of follicular atresia [14]. Investigations into follicular atresia primarily examine the molecular regulations of GC apoptosis that are proportional to ovarian response, with high apoptosis resulting in lower oocyte yield, lower numbers of MII oocytes and cleavage, as well as reduced blastocyst formation [18].

The two basic pathways of cell death include apoptosis and necrosis; the former describes programmed cell death while the latter describes “accidental” cell death [19]. Apoptosis is a tightly controlled process whereby damaged cells are removed and regulates cell numbers and tissue homeostasis in early development through to adult life [20], which underlies all functions of ovarian development, folliculogenesis, embryogenesis, as well as tumorigenesis [19]. Apoptosis serves both beneficial and detrimental effects, with GCs apoptosis required for normal development and a positive indicator of fertility [21]. It is also beneficial during embryogenesis to eliminate damaged cells and to allow for penetration of the embryo into the uterine lining [22]. On the other hand, excessive apoptosis may contribute to the pathogenesis of neurodegenerative diseases, the oncogenesis of malignancies, and female infertility [23]. An essential mechanism for apoptosis is proteolysis, and the caspase family of proteases is the key player involved [20]. These are highly conserved enzymes with a high affinity for aspartic acid, which they cleave to execute cell shrinkage, plasma membrane blebbing, and disintegration [19]. Initiator caspases (Caspase-8, -9, and -10) form the apoptosome complex, which turns on executioner caspases (Caspase-3 and -7) to dismantle cellular contents [24]. Caspase activation occurs late in apoptosis and is the ‘point of no return’ when the cells are irreversibly fated for death [25].

Apoptosis can be further divided into the extrinsic and the intrinsic mitochondrial pathways, both of which ultimately lead to caspase activation [19]. The extrinsic pathway involves the initiator caspase-8 and -10 [19] while the intrinsic apoptotic pathway regulates the balance between proapoptotic and antiapoptotic factors [B-cell lymphoma-2 (Bcl-2) family of proteins] [23,24]. Proapoptotic BAX can oligomerize and permeates the mitochondrial membrane [26], resulting in the leaking of cytochrome c, which activates initiator caspase-9 [23]. Antiapoptotic Bcl-2 stabilizes the mitochondrial membrane potential (MMP) by binding to and inhibiting BAX [8]. An additional class of Bcl-2 proteins can bind and inhibit Bcl-2; therefore, are considered proapoptotic regulators (e.g., BAD) [24]. The heat shock protein family (HSPs) is also involved in GC apoptosis and is associated with acute cellular responses to stress [27,28]. HSP70 is quickly induced by stress to hasten recovery and restore cell function. It inhibits apoptosis through the Akt kinase pathway where it regulates BAX [28]. Therefore, HSP70 is considered an antiapoptotic regulator.

Through bidirectional communication, GCs are crucial for proper oocyte maturation and development [8]. Consequently, apoptosis in GCs is linked to negative IVF outcomes and is shown to be higher in women with reproductive conditions such as polycystic ovarian syndrome (PCOS) [8,18]. Therefore, apoptosis in GCs speaks to the competence of the oocyte and follicle. Although apoptosis is required during crucial phases of folliculogenesis, it must be maintained under a threshold to ensure developmental competency [29]. BPA has been shown to induce apoptosis through the intrinsic mitochondrial apoptotic pathway in human placental cells [30], mice testes [31], human GCs [32,33], mice GCs [1], and bovine GCs [34]. This was evident with a decrease in MMP, induction of proapoptotic proteins, and activation of caspases. Mitochondrial function in oocytes is a strong indicator of embryonic developmental potential; reduced mitochondrial function results in a decline in fertilization, and increases in embryo DNA fragmentation and aneuploidy rates [33].

Functional studies have validated the involvement of miRNAs in follicular atresia, as miRNAs function as apoptotic mediators through posttranscriptional regulation of mRNAs [14]. miR-21, one of the most abundant miRNAs in GCs during oocyte maturation, is upregulated in cancers, in women with PCOS, and during follicular atresia [35,36]. It targets apoptotic transcripts PDCD4 to inhibit apoptosis and increase ovulation rates in GCs to prevent follicular atresia by promoting cell survival [14,37,38]. Overexpression of miR-21 in ovarian granulosa cells significantly inhibited apoptosis [39] and conversely, inhibition of miR-21 significantly increased apoptosis in mice, humans, and bovine GCs [38,40,41]. In addition, miR-21 regulates a key member of the intrinsic apoptotic pathway, Bcl-2 [[42], [43], [44]], suggesting a role in the intrinsic apoptotic pathway.

Previous studies have shown that BPA significantly increases antiapoptotic miR-21 expression in bovine oocytes and GCs [45]. Additionally, BPA is reported to induce apoptosis in GCs [3,4,46]. These paradoxical results appear to contradict each other; therefore, it is crucial to explore more thoroughly miR-21's involvement, or lack thereof, in BPA-induced apoptosis. Herein we investigate this relationship in vitro using bovine GCs cotreated with a miR-21 inhibitor and BPA followed by an analysis of the intrinsic mitochondrial apoptotic pathway, to strengthen our knowledge of miR-21-mediated regulation of apoptosis in a BPA toxicity model.

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