Background
The exiting of the follicles from the quiescent pool and their entry into the growing follicle cohort are accompanied by the proliferation of granulosa cells and increased serum levels of anti-Mullerian hormone (AMH). In other words, there is a direct relationship among primordial follicle pool, growing follicles and serum levels of AMH [
1]. Hence, serum AMH level measurement is known as a follicle growth marker and an assessment test for ovarian aging [
1]. Thus, the initial size of the primordial follicle pool is a major determinant of reproductive lifespan [
2]. As a member of the transforming growth factor-beta (TGF-β) family, AMH is produced postnatally by granulosa cells of primary follicles until large antral follicles develop in the ovarian tissue [
3].
The serum levels of AMH increase slowly until puberty, gradually decline in adulthood and reach the lowest levels in menopause [
3]. While AMH produced by the growing follicles locally inhibits primordial to primary follicle conversion, resulting in the maintenance of primordial follicle storage [
3], it has stimulatory effects on the growth of preantral follicles in primates [
4]. Therefore, it can be expected that higher serum levels of AMH lead to the exiting of fewer follicles (primordial) from the ovarian pool. The depletion of ovarian follicles is an age-dependent trend that begins from the middle of embryonic life, and it is expected to complete at the 74th year of life [
5]. When the number of follicles reaches about 1000, menopause begins [
6], which, in addition to the ending of women
’s reproductive life, is the beginning of unpleasant changes in the woman
’s physical and mental life [
7]. All of these events are due to severe hormonal changes [
7].
The improving potential of several herbs on ovarian function has been reported in various studies. Among different plants, two medicinal herbs, fennel, and flaxseed are reported to be effective in maintaining the ovarian follicular reserve (OFR) due to their antioxidant and phytoestrogenic properties [
8‐
11].
Fennel (
Foeniculum vulgare, FV), one of the oldest medicinal herbs, shows a wide range of clinical functions, including anti-inflammatory and anti-cancer activities, as well as estrogenic effects. The antioxidant effect of the ethanolic extract of fennel on female mice has also been confirmed [
12,
13]. This biennial plant could inhibit free radicals due to the high content of antioxidant compounds such as polyphenols and flavonoids [
10]. Our recent study indicated that fennel extract and seed administration (500 and 1000 mg/kg/BW) in pregnancy and lactation periods could improve F1 folliculogenesis, fennel extract at a dose of 500 mg/kg showed a more pronounced effect [
14]. Another research also showed that alcoholic extract of fennel (100
_200 mg/kg) could improve folliculogenesis due to its estrogenic property [
15]. Moreover, fennel extract alone and in combination with flaxseed could improve offspring OFR at PND1, PND56 and PND240 because of antioxidant and anti-apoptotic properties [
11]. The protective effect of fennel on the ovaries of cyclophosphamide-treated mice was also demonstrated by Hassanpour et al. [
9].
Flaxseed (
Linum usitatissimum, LU) is a rich source of lignans with a wide range of biological activity including antioxidant, antitumor, and weak estrogenic and anti-estrogenic effects [
16,
17]. The strengthening of the body’s antioxidant capacity using flaxseed is attributed to its high cysteine and methionine content [
18]. In females, its dose and duration of intake have relatively different effects on the reproductive system. Treatment of animals with 300 mg/kg body weight of LU for 25 days increased body, ovary, and uterus weight and led to precocious puberty, whereas at higher doses, it induced negative feedback [
19].
Keeping in mind that serum AMH assay is an approved method for predicting primordial follicle pool size and ovarian aging in mice and women [
20] and the complicated effects of FV and LU on the adult female reproductive system, the current study was aimed to investigate the effect of hydroalcoholic extracts of FV, LU, and their combination on the AMH expression at the cellular, molecular and hormonal levels as a follicle growth marker in the ovarian tissue of first-generation female mice pups.
Discussion
Considering that AMH is known as an inhibitor of new primordial follicle recruitment [
28], an indicator of ovarian aging, and preserver of primordial follicle stock, the main objective of the present study was to evaluate the effect of two known medicinal herbs (fennel and flaxseed) on serum AMH and ovary expression in first-generation female mice pups. Briefly, our data indicated that fennel significantly increased serum and ovary expression level of AMH.
There is ample evidence for the biological effects of AMH on the ovary. The expression of 707 ovarian genes is under the influence of AMH [
29]. Given the fact that granulosa cells postnatally produce AMH, primordial follicles do not express AMH. When squamous cells of the primordial follicle convert to cuboid cells, AMH expression begins [
28,
30]. AMH expression begins and rises in growing follicles, which in addition to the stimulatory effect on the growth of pre-antral follicles in primates [
4] prevents the recruitment of primordial follicles from the quiescent pool [
3]. The 120-day travel of follicles from the reservoir to ovulation occurs in two stages: gonadotropin-independent (initial, early, paracrine control) and gonadotropin-dependent (cyclic, late, endocrine control), with initial recruitment being regulated by AMH and the cyclic phase by FSH [
31]. The direct survey of primordial follicle storage and predicting female genital reproductive lifespan and ovary aging, chance of pregnancy and other clinical instances is not possible. Several methods such as ultrasonography, measurement of serum level of estradiol, inhibin B, FSH, and AMH are used, among which AMH is the most reliable [
27] because serum level of estradiol, inhibin B, and FSH are gonadotrophin-dependent (under the influence of the hypothalamus-pituitary-gonadal axis) and their production occurs in the late stage [
32,
33]. Developing follicles up to 4 mm (primary, secondary, pre-antral and antral follicles) show the highest expression of AMH [
26]. Our data were in agreement with these findings. Although the number of growing follicles of the fennel-treated group was not significantly higher than that of the flaxseed-treated group, fennel treatment significantly increased the number of antral follicles (highest AMH-expressing follicles). It seems that FV promoted follicle growth because more antral follicles were observed on PND240 after both FV and FV + LU treatment. This change was associated with a significant increase in the serum level of AMH and ovarian tissue AMH expression in the fennel-treated group. Our data are consistent with evidence for AMH inducing pre-antral follicle growth to the antral, stage as discussed above, which provides valuable evidence on AMH stimulatory effects in mice. Also, fennel treatment brought AMH expression closer to the control group. Even though the ovarian expression of AMH in the control group on PND240 was higher than it was on PND56, the serum levels of AMH were also slightly higher on PND240 than they were on PND56; however, this difference was not significant due to unknown reasons. Because the method of assessing serum level and AMH expression was the same in all groups, the reason for the proximity of these values at the two ends of this period is unknown.
Primordial to primary follicle transition is regulated by stimulants PDGF (platelet-derived growth factor) [
34], bFGF (basic fibroblast growth factor) [
35], LIF (leukemia inhibitor factor) [
36], KITL (kit ligand) [
37], BMP4, BMP7 (bone morphogen protein) [
38] and inhibitors AMH & SDF1 (stromal cell-derived factor 1) [
30,
39] from three sources: oocyte (SDF1, PDGF), granulosa cells (AMH, LIF, KITL), and interstitial cells (BMP4, BMP7, FGF7). The dose-dependent modulating action of AMH in interplay with the stimulants and inhibitors of primordial follicles recruitment ultimately leads to the prevention of their entry into the growing follicles and retention of a greater number of primordial follicles in the ovarian reservoir. Another study showed that AMH-treated ovaries contained 40% fewer growing follicles [
3] and AMH knockout mice had fewer primordial follicles [
30]. While the onset of primordial follicle stock formation begins in the fetal period, it ends before birth in humans and immediately after birth in rodents such as mice, and some of them remain quiescent for years in humans and months in mice [
40].
Regardless of the controversy surrounding the therapeutic benefit of medicinal herbs, they have attracted many people as an alternative treatment for controlling disease, because of increasing evidence of their effectiveness. The efficacy of medicinal plants can be attributed to a wide range of clinical properties, such as their anti-inflammatory, anti-cancer, and antioxidant effects [
13,
41]. Therefore, the question of the present study was to evaluate the effect of fennel and flaxseed treatment during intrauterine life and lactation on serum levels and ovarian expression of AMH-gene and protein on post-natal day 56 (puberty onset) and 240 (menopause onset). To the best of our knowledge, no study has studied this so far. Our data indicated that fennel consumption alone and in combination with flaxseed could protect the ovarian primordial follicle storage through increasing the serum level of AMH and also tissue expression of AMH- gene and protein while consumption of flaxseed alone decreased these values. These findings are in line with our previous study, which confirmed the protective effects of fennel and flaxseed on OFR due to phenolic contents of these plants that have estrogenic and antioxidant activity. We also assessed the level of Rutin as flavonoid content in these extracts so that, high level of flavonoid content was observed in the fennel hydroalcoholic extract [
11]. Probably the data of the current study are related to valuable content of flavonoids in the fennel extract. Although the anti-apoptotic effect of flaxseed on the granulosa cells has been reported earlier [
42], our publishing data revealed that compared to flaxseed, fennel showed a more significant anti-apoptotic effect to protect ovarian follicles. As previously demonstrated, fennel treatment could protect ovarian follicles against the side effects of cyclophosphamide [
9]. Fennel extract could induce folliculogenesis in mice and increase the number of growing follicles because of its estrogenic property [
15]. The pharmacological effects of fennel on many organs have recently been highlighted in a review article [
13]. Other research data showed the beneficial effect of fennel on kidney function [
43]. Although our study results did not confirm the effectiveness of flaxseed on OFR compared to fennel, a report in 2018 indicated the ameliorative effect of flaxseed in polycystic rats [
44].
Based on our data and previous studies, an increase in the amount of growth hormone reflects, on the one hand, the volume of follicles secreting it, and on the other, the greater inhibition of the release of primordial follicles from the ovarian stock.
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