Background
Obesity is a chronic and multifactorial disease whose etiology stems mainly from the imbalance between daily energetic consumption and usage with basal metabolism, although genetic and environmental factors are also involved. According to recent estimates there were 1.9 billion overweight individuals over 17 years old in 2014, representing 39% of the world’s adult population while 42 million children under 5 years of age were overweight or obese in 2013 (for review see [
1]). These data gain particular importance especially because obesity enhances cardiovascular risk factors, type II diabetes, neurological diseases, cancer and metabolic disorders [
1], as well as fertility and reproductive disorders in females [
2].
The ovulatory cycle is maintained by the functional and temporal integration of hypothalamus-pituitary-gonads (HPG) axis. The ovulatory function begins with pulsatile secretion of gonadotropin releasing hormone (GnRH) through the hypothalamus. GnRH stimulates the pituitary to release two important gonadotropic hormones: follicle stimulating hormone (FSH) and luteinizing hormone (LH), which act on granulosa and theca cells in the ovary by stimulating the synthesis of estrogens and androgens, respectively. In obese women, elevated serum levels of insulin and leptin cause disruption of this axis via ovarian and hypothalamic actions of these hormones [
3]. Insulin directly stimulates androgen synthesis in the ovaries, acting in synergy with LH to increase the production of androgens in theca cells, being considered a co-gonadotrophin [
4]. In turn, leptin stimulates neurons producing kisspeptin, a neuropeptide that enhances GnRH release [
5].
High evolutionary conservation of reproductive function between mammals allows a parallel to be drawn between rodents and humans [
6]. Rats with obesity induced by high fat diet presented early sexual maturity with greater follicular development, but with an accelerated rate of follicular loss and reduced fertile life. In addition, these animals presented more atretic follicles, suggesting that obesity may stimulate follicular apoptosis [
7]. Similar to rodents, clinical and epidemiological investigations have shown that excessive fat accumulation leads to irregular menstrual cycles and infertility [
8], while being associated with precocious puberty [
9] and polycystic ovary syndrome (PCOS) [
10].
Several drugs have been used to treat infertility disorders. Treatment of Wistar rats with clomiphene, an ovulation-inducing drug, improved the number of pups per litter [
11]. Additionally, it has been demonstrated in vitro that resveratrol inhibits the production of androgen by ovarian theca cells, being clinically relevant for conditions associated with hyperandrogenism, such as PCOS [
12], an endocrine disorder commonly associated with hyperinsulinemia and metabolic syndrome [
10]. Likewise, insulin-sensitizing agents have been used to reduce hyperinsulinemia, providing positive impacts on reproductive parameters [
13].
In this scenario, it is believed that medicinal plants are important sources of new chemicals with virtuous therapeutic properties and fewer side effects [
14]. Syzygium cumini (L.) Skeels (Myrtaceae), is a tree popularly known as jambolan in Brazil, jamun in India, black plum in Europe, among others, whose anti-diabetic effects have been widely studied, especially due to its low toxicity [
15]. Studies have indicated that ethyl acetic and methanolic extracts from
S. cumini seeds have anti-diabetic properties in rats with streptozotocin-induced diabetes [
16]. In addition to reducing glycemia, we showed that a polyphenol-rich extract prepared from
S. cumini leaves restored peripheral glucose tolerance while inducing insulin secretion [
17]. Of note, we have recently characterized the female L-monosodium glutamate (MSG) obese rat as a novel model to investigate the reproductive repercussions of obesity without interference of HPG axis [
18].
Thus, taking into consideration the epidemiological importance of obesity and its repercussions on female reproductive system, in the present study we sought to expand the applicability of a hydroethanolic extract of S. cumini leaves (HESc) to female reproduction disorder induced by the MSG obesity model. Additionally, by using MSG-obese rats, we were also able to assess the individual impact of obesity on reproductive function without a functional HPG axis, shedding new evidence on the inter-regulation of obesity on ovarian function.
Discussion
Female obesity leads to HPG axis disorders, resulting in reduced oocyte quality, endometrial receptivity and infertility [
24]. Thus, therapeutic measures of adiposity reduction and improvement of the metabolic profile have shown a positive correlation with recovery of ovarian morphology and reproductive health [
25]. At the same time, up until now there was no experimental model to address metabolic dysfunction without contribution of the HPG. In the present study, we have demonstrated that administration of HESc to obese MSG rats reduced total and periovarian adiposity, while restoring serum glucose and cholesterol levels with a relevant effect on glucose tolerance. These metabolic effects resulted in a significant recovery of ovarian follicle atresia, without improvement of estrous cycle, thus providing for the first time evidence that metabolic improvement have a positive impact on intraovarian environment regardless of HPG axis.
We have previously shown that neonatal MSG administration leads to a PCOS-like phenotype in young adult female rats [
18]. Additionally, we and others have demonstrated the beneficial effects of HESc on metabolic parameters of obese and diabetic animals [
17,
26]. Therefore, MSG-Syz animals displayed improved metabolic features, with reduced body weight, Lee index and fat accumulation. Such effects have been extensively studied and ascribed to the flavonoid content of HESc, specifically to myricetin and quercetin identified on this extract [
17,
26,
27]. In a study with onion hydroethanolic extract rich in quercetin, Moon et al. (2013) attributed to this flavonoid the antiobesity effects observed in rats fed a high fat diet, since there was suppression of preadipocyte differentiation and inhibition of adipogenesis via modulation of β-oxidation of fatty acids, thermogenesis and lipid metabolism [
28]. In this way, we can suggest that the antiobesity effect observed here is likely due to the flavonoid constituents present in HESc.
HESc displayed an anti-hyperglycaemic effect, reducing glycemia levels by 15% in MSG-Syz rats, bringing it back to CTRL levels. Moreover, HESc completely restored peripheral glucose tolerance. Such antidiabetic effects are in agreement with previous literature on the matter. For instance, Anandharajan et al. (2006) demonstrated that methanolic extract of
S. cumini increased the expression of glucose transporter type 4 (GLUT-4) in a PI-3-kinase-dependent manner, promoting the activation of peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway - an effect correlated with increased GLUT-4 transcription and consequent uptake of glucose [
29]. This mechanism is of paramount importance to the obesity model under study, since obese MSG rats have a reduced number of GLUT-4 transporters in insulin-sensitive tissues [
30]. Added to this, another report showed that the hydroethanolic extract of
S. cumini leaves improved superoxide dismutase activity in different tissues of diet-induced obese mice, attributing such therapeutic effect to the antioxidant properties of the extract evaluated [
26]. Nonetheless, we have shown that the same extract used on the present study induces insulin secretion both in INS-1E pancreatic β cells and ex vivo islets [
17] without significant toxicity in vitro (data not published). Thus, we can corroborate previous literature and infer that the significant improvement of glucose metabolism found on MSG-Syz rats is likely due to the above-mentioned mechanisms.
In addition to the effects on glucose metabolism, administration of HESc to MSG rats resulted in a significant reduction in serum cholesterol levels. The hypolipidemic effect of
S. cumini has been evidenced in works carried out with different extracts of the seeds and fruits of this species, which have attributed this action to the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase enzyme, a key enzyme of biosynthesis cholesterol [
31,
32]. Therefore, the marked reduction on cholesterol levels seen in MSG-obese female rats is in agreement with previous literature, providing evidence not only that HESc effects are consistent in both genders but also advocating the use of female animals for preclinical studies of hypolipemiant drugs.
Obesity is commonly associated with dysfunctional HPG axis, causing increase on testosterone and LH levels, mainly due to hyperinsulinemia [
4,
33]. In fact, most PCOS models display hyperandrogenism, with increased LH levels [
34]. On the other hand, female MSG rats do not follow such pattern, because the neonatal injection of this chemical severely damages the median eminence and arcuate nucleus of hypothalamus, leading to an obese animal with low levels of growth hormone (GH) as well as all of the above mentioned HPG hormones [
18,
35,
36]. In fact, we described this model as a cheap and feasible tool to investigate the impacts of metabolic syndrome on female reproduction without the interference of HPG axis [
18]. Corroborating our previous data, MSG animals displayed unaltered sex hormones, while MSG-Syz treatment was not sufficient to effectively affect these. This could also explain the lack of effect HESc had on estrous cycle, given that the HPG axis is the main regulator of female cycle. Surprisingly, though, HESc administration improved follicular count with recovery of follicular atresia shown in primary and antral follicles of MSG-Syz animals. While other flavonoid-rich extracts have shown positive effects on metabolic disorders, to the best of our knowledge this is the first description of HESc having a positive impact on ovarian function. Likewise, due to the experimental model used in other studies, most interventions result in recovery of both estrous cycle, HPG axis and ovarian follicle health [
37,
38], making this the first report to show a detachment between these features, possibly due to the unique characteristics found on MSG rats. Whether the positive effect of HESc on intraovarian environment is an early outcome that would predict improved reproductive function or an isolated effect without further consequences is yet to be established, being a limitation of the present report.
Periovarian adipose tissue has received increasing attention over the past few years. In fact, Wang et al. [
39] recently described the importance of ovary fat pad to reproduction in lean mice. Likewise, several reports relate the hypertrophy of periovarian adipocytes to ovarian dysfunction in different models [
40,
41]. Therefore, given the beneficial effect of HESc on ovarian follicle development, we sought to determine whether periovarian fat pad is in some way related to the abovementioned effects. MSG rats once again corroborated previous data, showing hypertrophied periovarian adipocytes, whereas MSG-Syz had adipocytes’ comparable to CTRL. It seems reasonably to suggest that the paracrine effects exerted by periovarian adipocytes are important to the ovarian microenvironment and the positive effect seen on ovarian follicles from MSG-Syz could be related to the reduction on periovarian adipocyte area. Such hypothesis should be further investigated in future works.
Conclusions
As a whole, this work shows for the first time that the hydroethanolic extract of S. cumini leaves produces beneficial effects on the metabolic parameters of female MSG-obese rats, without improving their oligocyclicity. Even so, HESc administration improved ovarian follicle health – an effect in some measure due to reduction of periovarian adipocytes. Not less important, this is the first report to show the PCOS-like features of MSG-obese rats can be at least partially reversed by pharmacological treatment, providing novel evidence that, without a functioning HPG axis, metabolic improvement is ineffective for estrous cyclicity, but critical for follicle health. Future perspectives include longer or earlier treatments, which may result in greater benefits on reproductive parameters of MSG-obese female rats.