Obesity is a multifactorial disease whose contributing factors include genetic predisposition and ingestion of foods having a high fat content. Such excesses combined with insufficient physical activity promote development of obesity while the genetic influence affects individual susceptibility to dietary intake [31]. Typical characteristics of obesity include excessive body mass and excessive intake of fat laden nutrients that are stored rather than metabolized for energetic needs.
In humans, the definition of a HFD includes plans in which 30–70% of their caloric value is derived from fats [32]. In order to simulate the conditions leading to human obesity, we generated the female obese HFD mouse model by providing a diet in which fats provided 60% of the caloric value whereas in the ND control group it was reduced to 10%. The results showed that both the body weight and abdominal fat mass increased in the HFD fed mice relative to those fed on a ND. These results are somewhat consistent with a previous report showing that adipocyte volume expansion and their abundance can both increase as a consequence of excessive food consumption [33]. Such rises occur because adipocytes function as fat depots [33]. In this study, we also verified that the HFD is a relevant obesity model because it led to fat store accumulation in the female reproductive system. This observation are in agreement with increases in serum lipid profiles involving rises in total CHOL, HDL and LDL. On the other hand, the liver is the major organ for lipid metabolism and lipid accumulation reaches excessive levels on a HFD. These increases can definitely damage its morphological structure and function, finally leading to many hepatic diseases such as fatty liver and hepatocirrhosis. Moreover, many fat vacuoles appeared in the hepatocytes and a severe hepatic steatosis developed in mice fed the HFD indicating establishment of a relevant HFD model.
Recently, it has become apparent that there are many life threatening diseases associated with obesity, including diabetes mellitus, cardiovascular diseases, cancers, hepatic dysfunction, etc. [2]. In general, female obese patients frequently suffer from several kinds of gynecological disorders such as endocrine dyscrasia, polycystic ovary syndrome (PCOS), amenorrhoea and even infertility [2, 3, 6]. Our results of breeding trials showed that the number of pups delivered by obese mice was significantly reduced in the HFD fed group relative to those on the ND. Due to the known detrimental effects of obesity on female fertility, we characterized its harmful effects on the female reproductive organ morphology. In the HFD group, huge fat droplets accumulated in the stromal layers of the uterus, ovary and oviducts and their histological integrity was extensively altered. The changes included hyperproliferative uterus, vacuolated ovarian tissue and thickened oviduct walls.
In our study, obesity-induced damage to the female reproductive system is associated with increases in IL-6 and TNF-α mRNA and protein expression levels based on an agreement in the results of IHC staining, Western blotting and quantitative RT-PCR in the HFD female reproductive organs. Such increases were relevant to MAPK signaling activation and increases in NF-κB expression. Many previous studies reported that adipose tissue acts as a dynamic endocrine organ, which can secrete numerous pro-inflammatory cytokines such as IL-6 and TNF-α [4]. On the other hand, obesity was recently recognized as a low-grade chronic inflammatory status [33], and IL-6 and TNF-α are considered as biomarkers of this condition. These cytokines have pivotal roles in mediating inflammation, hematopoiesis, cell proliferation and apoptosis [34–36]. Accordingly, our results indicate that mice fed a HFD acquire a chronic inflammatory status during obesity development. This outcome shows that the HFD model is relevant to delineating how infertility develops in the human obese population [12, 25, 37, 38], because in obese human females fat accumulates in adipocytes and other organs or tissues such as liver, smooth and skeletal muscles [39].
The ERK1/2, p38 and JNK signaling pathway control can become maladaptive inducing responses associated with a wide range of diseases including cancers, ischemic heart disease, autoimmune diseases, etc. [40]. Different cytokines or growth factors interacting with their cognate receptors mediate control of cell proliferation and differentiation, inflammation through modulating either cell cycle progression or transcription factors suppressing tumor formation [41, 42]. In females, this activated pathway induced by inositol can lead to endothelial dysfunction in preeclampsia [43] and it also plays a critical role in the pathogenesis of PCOS, but it decreases the steroidogenic response to gonadotropins in preovulatory granulosa cells [44]. On the other hand, ERK1/2 inhibition along with activation of either p38 and/or JNK can lead to increases in inflammation and atherosclerosis.
p38 and JNK can be activated by various stress stimuli such as UV, which induce apoptosis [45]. p38 is an oxidative stress-response kinase and its role in the female reproductive organs is quite complex during the entire pregnancy process and parturition. p38 MAPK is relevant to some processes such as decidualization, trophoblast differentiation and invasion [46], myometrial quiescence or activation during parturition, and placental growth [47]. Pathologic activation of the p38 pathway can cause adverse pregnancy outcomes including preterm birth [48]. Maladaptive JNK signal pathway activation is also associated with numerous female reproductive diseases. For example, solely inducing sustained JNK/AP-1 signaling pathway activation is sufficient to induce delivery, however, LPS-induced rises in TNF-α expression levels lead to inappropriate JNK pathway activation, which usually results in premature delivery [49]. Besides, activated JNK induced by oxidative stress is also associated with granulosa cell apoptosis [50].
It is known that maladaptive MAPKs signaling pathway activation induces adipogenesis. However, the involvement of the role of the ERK1/2 signaling pathway in this process is somewhat controversial. Some studies claimed that this pathway instead inhibits adipogenesis [51], whereas others suggested that it promotes adipogenesis [52]. Moreover, some reports indicated that this pathway promotes adipogenesis in the initial stage whereas in the later stages it has a negative role in this process [53]. Additionally, a JNK1 deficient mouse is reported to contribute to obesity development suggesting lack of JNK1 involvement in this process [54, 55] and p38 signaling pathway activation can enhance the adipogenesis [56, 57]. Taken together, there is substantive evidence that the MAPK signaling pathway activation by various stressors contributes to inducing obesity by enhancing adipogenesis.
NF-kB is a transcription factor mediating signaling pathway control of a myriad responses in health and disease. This transcription factor undergoes activation by hyperlipidemia in obese patients. It regulates the expression of immediate-early response genes involved in stress and inflammation and contributes to various female reproductive diseases [58]. NF-kB activation normally occurs in the myometrium prior to delivery and untimely activated NF-kB leads to premature delivery [59]. More importantly, some studies elucidated crosstalk of NF-kB with other pathways in the pathogenesis of ovarian cancer [60].
Several alternative mechanisms could account for how the HFD induced increases in MAPK signaling and upregulated NF-κB expression which led to adipocyte hypertrophy and hyperplasia in the female reproductive system. a) Direct activation by IL-6 and TNFα of MAPK signaling and upregulation of NF-κB expression. IL-6 could induce such responses via directly activating many signaling pathways such as NF-kB, MAPKs, PI3K, mTOR and AMPK [36]. b) Direct activation by IL-6 and TNFα of their cognate receptors, which in turn increases MAPK signaling and upregulates NF-κB expression. For example, rises in TNF-α gene and protein expression levels stemming from the altered lipid profile in pathological adipogenesis could induce increases in MAPK signaling and upregulate NF-κB expression through interacting with its cognate receptors, TNFR 1 or TNFR2 [61]. c) One or more of the constituents in the altered lipid profile may stimulate non-cognate receptors and subsequently increase MAPK signaling and NF-κB expression. All three of these possibilities are tenable since in different tissues one or more of these three alternatives accounts for how adipogenesis increases IL-6 and TNFα expression levels.
Herein, it is evident that the increases in fat droplets are associated with rises in the IL-6 and TNF-α expression levels and damage to the reproductive organ structural features and functions of the fat laden uterus, ovary and oviduct. Any or all of these effects can contribute to reducing fertility and reproductive success. Furthermore, the activated NF-kB pathway is involved in inducing immune responses that can aggravate increases in the inflammatory status in the uterus, ovary and oviduct, whereas activation of the MAPKs signaling pathway augments adipogenesis in the female reproductive system (Fig. 9).