In utero and postnatal exposure to Foeniculum vulgare and Linum usitatissimum seed extracts: modifications of key enzymes involved in epigenetic regulation and estrogen receptors expression in the offspring’s ovaries of NMRI mice

The ovary is the main organ of female reproductive system. In utero development of this organ highly depends on the signaling pathways of endogenous estrogen hormone, hence developmental exposure to exogenous estrogens may disturb this process, and affect offspring’s endocrine system and ovarian function [1].

One of the main important group of the environmentally exogenous estrogens is phytoestrogens; dietary estrogens produced by plants that are structurally or functionally similar to endogenous estrogens. These substances are divided into four groups: lignan, stilbenz, isoflavonoids, and flavonoids [2]. They work by activating estrogen receptors in a variety of organs, including the ovary and uterus, to have effects that are comparable to those of endogenous estrogen [3]. Depending on the cell type, dose and time of exposure, they can have estrogenic or antiestrogenic effects [4]. The exposure effects of different phytoestrogens on the ovarian function, apoptosis and estrogen receptors (ESRs) expression of the female reproductive system were determined [5‐7]. Besides, regarding to the placental and milk transmission of phytoestrogens, these bioactive compounds might affect the later health status of the offspring. Various effects such as premature puberty, impaired ovarian folliculogenesis and estrous cycle as well as impaired anogenital distance were reported following prenatal and postnatal exposure to phytoestrogens [8, 9].

Among the medicinal plants, foeniculum vulgare, commonly known as fennel, and Linum usitatissimum, commonly known as flax, are prevalent plants in the Mediterranean region. They used in traditional medicine to woman’s health and treat a variety of diseases [10‐12]. Therefore, these plants are traditionally used to make Qavut in Iran (Kerman provinence) and fennel could increase breast milk production [13]. They are rich in phytoestrogens, as fennel contains a high concentration of phytoestrogenic compounds such as flavonoids included of eriodictyol, quercetin-3-rotinoside, and rosmarinic acid [14], and flax has highest level of lignans [15] among different medicinal plants. The positive effects of fennel and flaxseed exposure on ovarian function have yet been well determined [16‐18]. Furthermore, flax root lignans could alter the expression of ESRs in the breast cancer cells [19]. We have recently reported that pre and postnatal exposure of mice to hydroalcoholic extracts of both plants could alter the ovarian folliculogenesis, apoptotic genes expression and oxidative stress in first generation (F1) of female offspring [20‐22].

However, maternal lifestyle factors like diet [23, 24], especially during the time of development, may change how genes are expressed via epigenetic phenomena. Because these changes are independent of genomic sequencing alteration, they may have an impact on future health or disease risk [25, 26]. Three mechanisms of the epigenetic modifications are DNA methylation, histone modification, and regulation of non-coding RNAs. DNA methylation is performed by the DNA methyltransferases (DNMTs); DNMT1, DNMT3A, and DNMT3B. Also, histone modifications are done by histone acetyltransferase (HAT) and deacetylases (HDACs) enzymes. A previous study showed that pre and postnatal exposure to a phytoestrogen-rich diet containing genistein and daidzein can alter DNA methylation of the ESRα gene in the female offspring [27]. Such alterations could have long-term effects to prevent the ovarian and prostate cancers by modulating DNMTs and HDACs activities [28].

However, the overall safety, hazardous and beneficial levels of a dietary regimen supplemented with plants that are high in phytoestrogens on the long-term health of the offspring are still unknown, despite recent increases in research on the effects of phytoestrogens on health status. Therefore, regarding the potential of phytoestrogens in epigenetic alterations, for the first time, we aimed to examine the early-life exposure to fennel and flaxseed-based diets, which are very rich in natural phytoestrogens, on the expression level of enzymes involved in the epigenetic modifications of the ovaries in adulthood.

Despite the similar biological activity of phytoestrogens as the endogenous estrogen on ESRs and the importance of signaling pathways of ESRs in the differentiation and maintenance of female reproductive tissues, so far, no study has surveyed the effect of perinatal exposure with lifelong exposure to hydroalcoholic extracts of these plants on the expression level of the ovarian ESRs in adulthood, that’s what we evaluated in the current study.

This in vivo study is the first to reveal that prenatal and postnatal exposure to the fennel and flaxseed extracts could lead to F1 ovarian epigenetic changes via differential expression of DNMTs and HDACs as well as ESRs. These changes might affect the normal function of the ovary in adulthood.

Complex molecular regulations during in utero development are a result of interactions between genetic and epigenetic. Maternal lifestyle [23, 24], such as a dietary supplement with phytoestrogens is one of the environmentally epigenetic effect elements that could affect developmental programming processes [26]. However, maternal steroid hormone-binding protein (SHBG) and fetal α-fetoprotein are the main modifiers of fetal exposure to phytoestrogens that may protect the fetus partly from the destructive effects of exogenous estrogens such as phytoestrogens [31]. Besides, breast milk could be another transporter pathway for infants' phytoestrogen exposure. Because the reproductive system and germ cells are developing at these critical times, phytoestrogens may act as exogenous estrogens, making them particularly vulnerable. To date, several studies extensively determined [1] that early developmental exposure to environmental estrogens could reduce follicular growth and primordial follicle numbers and alter follicle assembly, resulting in the formation of ovarian cysts and multi-oocyte follicles [31], therefore would lead to lower concise reproductive lifespan. Nevertheless, although there is limited information on ovarian epigenetic changes after individual exposure [32], to date, there is no evidence regarding ovarian epigenome alteration following long-term exposure to phytoestrogens.

One of the main underlying mechanisms involved in epigenetic modifications is the alteration of the DNA methylation status of specific genes. In general, DNA methylation was occurring by two types of DNMT; de novo (DNMT 3A and 3B) and maintenance (DNMT1) [33]. Our data showed an overall decrease of DNMTs expression in the ovarian tissue of all exposed F1 mice, especially in the fennel exposed mice. Recently, Harlid et al. [34] reported that neonatal exposure to soy’s phytoestrogens in the formula could lead to alteration of DNA methylation level in vaginal epithelial cells, which may be related with reduced estrogen-responsive genes and probably later-life health problems. Besides, phytoestrogens are sometimes referred to as endocrine disruptors. There are several reports that early life exposure to several endocrine disruptors with hormone-like activity, such as methoxychlor (MXC) [35], bisphenol A (BPA) [36], and diethylhexyl phthalate (DEHP) [37] could change ovarian DNA methylation. Abnormal methylation is potentially related to differential individual vulnerability to the later development of disease, such as various cancers [38, 39]. It was shown that overexpression of DNMTs could cause hypermethylation of tumor suppressor genes resulting in cell transformation in different cancers [40, 41]. In addition to DNA methylation, histone aberrant acetylation pattern is also associated with the modification of epigenetic signature. HDACs family members are involved in the suppression of DNA transcription by removing the acetyl group from histones. A raise in HDACs was reported in cell proliferation and invasion through repression of tumor suppressor genes [42, 43]. To date, 18 different mammalian HDACs have been categorized into four classes [44, 45]. Class I including HDAC 1, 2, 3, and 8 are mostly involved in carcinogenesis compared to others [46]. It has been revealed that there was a decrease of DNMTs (DNMT1, DNMT 3A, and DNMT3B) as well as HDAC2 expression levels in the mammary gland of F1 rats that underwent a phytoestrogen-enriched regimen during in utero development, breastfeeding, and puberty periods [47]. Also, one previous study reported decreased expression levels of DNMT1, DNMT3A, DNMT3B as well as HDAC1 and HDAC2 in the uterus of adult offspring following neonatal genistein exposure [48]. In the present study, the downregulations of HDAC1 and HDAC2 were observed in the ovary of adult mice that were exposed to either fennel or flaxseed extracts. It is well established that HDACs have a physiological role in cell proliferation and survival, so that overexpression of HDACs I and II has been seen in the various tumor cells [49], including ovarian cancer tissue [50]. Hence, HDAC inhibitors have been used as tumor therapeutic agents by inducing apoptosis [51]. Nowadays, there is special attention to phytoestrogens for cancer therapy because of their effects on reducing the HDACs expression and also activity [52, 53], although some reports reported no marked effects [54]. Different used doses and durations may explain the contradictory results [55]. Our findings suggest that each of these plants may function as an HDAC expression down-regulator in the ovary, and as a result, they may be considered an element that protects the ovary by preventing abnormal cell growth and cell cycle progression, likely through hypomethylation and histone deacetylation of tumor suppressor genes. A decreased growth of renal cancer cell lines via decreasing of DNMTs and HDACs activity was seen following exposure to the genistein phytoestrogen in a dose-dependently manner [56]. However, similar to DNMTs, reduction of HDACs transcriptions was pronounced in the pups that were exposed to the fennel compared to the flaxseed. Hence, ovarian epigenome alteration was more affected by fennel rather than flaxseed.

On the one hand, one of the target sites for these epigenetic enzymes' function is the promoter region of the estrogen receptors; ESRα and ESRβ. Both isoforms are involved in the ovarian development, by triggering the estrogen signaling pathway. Similar to the endogenous 17-beta estradiol, and competitively affinity, phytoestrogens act on these receptors, although ESRβ is a selective receptor for them, therefore exposure to these plant-derived estrogenic compounds could affect reproductive biology, development and physiology. It was shown that early-life exposure to several chemicals with hormone-like activity results in long-term changes in the ESRβ gene in the ovary. Altered ESRs activity might lead to changed ovarian folliculogenesis and steroidogenesis. However, different ESRs expressions could be because of the modification of the epigenetic mechanisms that regulate it. DNA methylation of promotor regions of genes by DNMTs, which usually results in gene expression suppression, is one of them. Several reports showed endocrine disruptors could lead to high expression levels of DNMTs in the ovary. Changes in ovarian DNMT expression can disrupt the expression level of many transcription factors, which regulate the gene expression associated with the hormone action, paracrine growth factors, and extrinsic innervation. For example, MXC could induce DNMT and hypermethylation of ESRβ promoter region in the ovary as a result lead to a decrease of ESRβ protein, lower response to endogenous circulating estradiol (E2) and the alteration of E2-dependent follicular development in adulthood. Therefore, a relationship between decreased ESRβ expression and increased DNMT expression was determined. However, as yet, such a relationship was not studied in the adult ovary following long-term exposure to phytoestrogens, which is evaluated in the current study. Our data for the first showed a differential expression of ESRs, along with the epigenetic changes in one direction, in the ovarian tissues of all pups that were exposed to fennel and flaxseed. Nevertheless, evaluating of the methylation level of ESR promoters along with the changes in the expression level of epigenetic modification enzymes may provide more complementary information. Previous studies [35, 57] reported that pre and neonatal exposure to some endocrine disruptors could cause ovarian dysfunction via a reduction of ESRβ expression because of DNMT3B overexpression and hypermethylation of the ESRβ promoter region that finally leads to a lesser response to endogenous estrogen.

However, regarding the reduced expression level of both ESRs, fennel and flax, alone may act as agonists leading to ESRs down-regulation following their long-term exposure. This decrease could cause a lower response to E2 in adulthood and change the folliculogenesis regulated by E2 and several paracrine factors such as anti- mullerian hormone (AMH). As reported in our previous study [22], the serum estradiol level was highest in the fennel exposed mice and lowest in the flax-received mice, while animals that received the fennel and flax seed concomitantly had a lower level rather than fennel. An invert relationship [58], between circulating E2 level and ESRs mRNA Level was reported as ovariectomized animals had a lowest level of E2 along with the increasing ESRs mRNA and treatment with E2 led to increasing circulating E2 along with decrease of ESRs expression level. In agreement with them, we found a high level of circulating E2 in the fennel group while exhibited a sharp reduction of ESRs expression. The flaxseed-exposed mice with low circulating E2 had higher level of ESRs mRNA in compared to the fennel and flax + fennel-exposed mice. Besides, in the combined group, the circulating E2 was less than fennel group and ESR beta mRNA was higher than fennel-exposed mice and ESR alpha was very greater than fennel and flaxseed-exposed mice.

It is well showed that the function of these isoforms of ESRs is different so that almost, anti-proliferative effects are related to the ERβ and proliferative effects are related to the ERα. Ultimately, the ratio of ESRα-to β could determine the net effect of these plant-derived compounds on the target tissue. A high ratio may associate with cellular proliferation stimulation, while the opposite effect was seen at a low ratio. A previous study showed that a low dose of biochanin A; a type of dietary phytoestrogens could stimulate the growth of human mammary carcinoma cells by increasing of ESRs expression level while exhibiting the opposite effect at high dose [59]. However, phytoestrogens have the potential to alter ESR conformation, leading to partial agonist and antagonist properties dependent on the target location and their dose. Based on our data, these extracts may be potential candidates for finding herbal chemo-protective drugs via the overall reduction of the ESRα to β ratio, although more research is needed to test this issue. Surprisingly, those animals that were exposed to a mixture of fennel and flaxseed extracts showed a rise in the mRNA expression of ESRα along with ESRβ reduction. Considering that the overall ratio is toward increasing the ESRα/β ratio, therefore exposure to the very high levels of phytoestrogens in a mixture of these plants might lead to re-stimulation of growth and division of cells, which is seen in almost cancers, such as breast cancer. However, we observed overexpression of HDAC2 along with ESRα in the ovaries of pups that were exposed to both extracts, simultaneously, which is in disagreement with some studies that determined the high activity of HDACs are related to estrogen receptor suppression [49]. Regarding increasing of ESRα /β ratio [60] as well as overexpression of HDAC2 [46, 61, 62] that have been reported in several tumor cells, such as ovarian cancer, our data make us more cautious in the long-lasting exposure to the high concentrations of complex phytoestrogens in the combination of these two plants. In line with our data, some animal studies reported that neonatal exposure to genistein could cause an enhanced risk of tumors [63]. However, as yet, there is no conclusive ground regarding the action of phytoestrogens as antitumor or tumor growth stimulation elements [64].

Therefore, our data clearly showed the changes of the ovarian epigenome. This alteration might affect the oocyte development by the alteration of the expression level of genes-involved in the secretion of ovarian metabolite, endocrine and paracrine factors, derived from ovarian somatic cells, that progress the oocyte maturation and leading to transgenerational phenotypes in reproductive health in the next generation that don’t exposed directly to this kind of endocrine disruptors. Thus, more studies are needed to determine whether ovarian epigenome changes could alter genes that interact to govern oocyte development in the developmental programming of the germline. Besides, because the uterus can provide an environment for developing the fetus, so the change of the intrauterine estrogenic environment can affect the reprogramming of the fetus, and lead to unfavorable reproductive outcomes, for example, exposure to high levels of androgens during intrauterine life can cause changes in the methylation of some specific genes, therefore it could cause polycystic ovarian syndrome in the children [65]. Consequently, ovarian epigenome changes might affect offspring’s reproductive system function either by damage to the growing oocyte that is exposed to different metabolites, paracrine/endocrine factors, or by the changed intrauterine estrogenic environment that interacts with the embryo's genetics. Reprogramming of developing germ cells may also be affected following these exposures that have transgenerational epigenetic impacts. Reducing the methylation status in oocyte’s imprinted genes is reported following exposure to DEHP and BPA as endocrine disruptors [66]. Also, a decreased methylation of histones in cultures follicles was reported following BPA exposure [67]. Therefore, more research is needed to elucidate the epigenetic modification of female gamete following exposure to phytoestrogens.

On the other hand, we observed a marked increase in the body, ovarian weights and coefficient of the fennel-treated mice. This increase of net weight could be a marker for estrogenic properties of the various kinds of phytoestrogens in the fennel extract, although this rise was not observed in the flax-exposed mice. Our findings are consistent with previous findings supporting that genistein phytoestrogen could increase the reduced total body and ovarian weights following radiation of rats in premature ovarian failure [6]. A previous study [5] showed that exposure of post-weaning female rats to genistein could reduce the weights of the ovary, uterus, and body at a low dose while showed reversed effects in high doses.

To conclude, our findings showed that pre and postnatal exposure to naturally-derived estrogens in a diet regimen supplemented with fennel or flaxseed extracts could lead to the alteration of ovarian epigenetic, as well as hormone receptor populations in the F1 adult pups. However, the full details of the underlying mechanisms of phytoestrogens' actions on the ovary have not yet been accurately defined and more studies are needed. Moreover, based on the different outcomes about the combination of these extracts compared to each extract, alone, it seems that it is still too early to introduce the safety of these plants as a portion of a diet regimen, especially during early life.